HK1083866B - Hla-a24-restricted cancer antigen peptides - Google Patents

Description

HLA-A24 restricted cancer antigen peptide

Technical Field

The present invention is in the field of cancer vaccine therapy. The present invention relates to HLA-a 24-restricted cancer antigen peptides, and more particularly, to HLA-a 24-restricted cancer antigen peptides derived from WT1 and having activity of inducing CTLs in vivo, polynucleotides encoding the same, cancer vaccines comprising the same, uses thereof as cancer vaccines, and methods for treating and preventing cancers based thereon.

Background

Cellular immunity, particularly cytotoxic T cells (hereinafter referred to as CTLs), plays an important role in eliminating cancer cells or virus-infected cells in vivo. CTLs recognize a complex formed between an antigen peptide derived from a cancer antigen protein on cancer cells (cancer antigen peptide) and MHC (major histocompatibility complex) class I antigen (referred to as HLA antigen in humans), and thus attack and destroy cancer cells.

In Immunity, vol.10: representative examples of cancer antigen proteins are listed in table 1 described in 281, 1999. Specific examples include melanosome antigens such as melanocyte tissue specific protein, gp100(J.Exp.Med., 179: 1005, 1994), MART-1(Proc.Natl.Acad.Sci.USA, 91: 3515, 1994), and tyrosinase (J.Exp.Med., 178: 489, 1993); and HER2-neu (j.exp.med., 181: 2109, 1995) and cancer markers such as CEA (j.natl.cancer inst., 87: 982, 1995) and PSA (j.natl.cancer inst., 89: 293, 1997) as cancer antigen proteins other than those from melanoma. Cancer antigen peptides are peptides consisting of about 8 to 11 amino acid residues, which are produced by processing cancer antigen proteins by intracellular proteases (cur. opin, immunol., 5: 709, 1993; cur. opin, immunol., 5: 719, 1993; Cell, 82: 13, 1995; immunol. rev., 146: 167, 1995). The cancer antigen peptides thus produced bind to MHC class I antigens (HLA antigens) to form complexes, and then the complexes are presented on the cell surface and recognized by the above CTLs. Therefore, in the development of cancer immunotherapeutic drugs (cancer vaccines) based on the destruction of cancer cells by CTLs, it is very important to identify cancer antigen peptides from cancer antigen proteins that can effectively induce CTLs.

In MHC class I molecules there are a number of subtypes and the amino acid sequences (binding motifs) of antigenic peptides bound to the respective subtypes follow a certain law. For the binding motif of HLA-A2, for example, the amino acid at position 2 is leucine, methionine, or isoleucine, and the amino acid at position 9 is valine, leucine, or isoleucine. For the binding motif of HLA-A24, the amino acid at position 2 is tyrosine, phenylalanine, methionine, or tryptophan, and the amino acid at position 9 is phenylalanine, leucine, isoleucine, tryptophan, or methionine. Recently, databases were searched (e.g., BIMAS software; http:// BIMAS. dcrt. nih. gov/molbio/HLA _ bind /) for any peptide sequence that includes the above-indicated motif and is expected to bind to HLA antigens. Therefore, in order to identify cancer antigen peptides capable of inducing CTLs from cancer antigen proteins, a peptide region consisting of about 8 to 11 amino acids in length matching a binding motif or a peptide sequence corresponding to a desired HLA type is first identified from the amino acid sequence of the cancer antigen protein.

However, peptides identified on the basis of the binding motif or expected peptide sequence are not necessarily immunogenic. Since the antigenic peptide is produced by intracellular processing of the cancer antigen protein, a peptide that is not produced by processing cannot be an antigenic peptide. Furthermore, since many cancer antigen proteins are originally present in a living body, even if peptides having a binding motif or a desired peptide sequence produced inside cells are used as cancer antigen peptides, CTLs are likely to be resistant to such cancer antigens. These facts indicate that, in order to identify cancer antigen peptides having activity of inducing CTLs, prediction based on only motifs of expected HLA types or desired peptide sequences is insufficient, and evaluation of immunogenicity (activity of inducing CTLs) in vivo is very important.

Based on the analysis of WAGR syndrome, which is a complication of Wilms ' cancer, aniridia, genitourinary abnormality, mental retardation, etc., Wilms ' cancer suppressor gene WT1(WT1 gene), which is one of Wilms ' cancer causative genes, was isolated from chromosome 11p13 (Nature, 343: 774, 1990). WT1 has a genomic DNA of about 50Kb and consists of 10 exons, and its cDNA has a length of about 3 Kb. The amino acid sequence deduced from the cDNA is shown in SEQ ID NO: 1(cell., 60: 509, 1990). The fact that the WT1 gene was highly expressed in human leukemia and that the cell growth of leukemia cells could be inhibited by treating leukemia cells with WT1 antisense oligomer suggests that the WT1 gene can promote the growth of leukemia cells (Japanese patent laid-open (Kokai) No. 104627/1997). Next, from the fact that WT1 is also highly expressed in solid cancers such as gastric cancer, colon cancer, lung cancer, breast cancer, embryonal cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer and ovarian cancer (Japanese patent publication (Kokai) No.104627/1997, Japanese patent publication (Kokai) No.35484/1999), it was also confirmed that WT1 gene is a neooncogenic protein for leukemia and solid cancers (J.Immunol., 164: 1873-80, 2000, J.Clin.Immunol., 20, 195-202, 2000). It is preferable to use a drug for cancer immunotherapy (cancer vaccine) for as many cancer patients as possible, and therefore it is important to identify cancer antigen peptides highly expressed in many types of cancer from WT1 and to develop a cancer vaccine based on these cancer antigen peptides. In the context of the present invention, WO00/06602 and WO00/18795 describe naturally occurring cancer antigen peptides which are composed of a portion of the WT1 protein.

In developing cancer vaccines, evaluation of in vivo efficacy of vaccines cannot be performed using a mouse of a pure line which is generally used as an experimental animal, but an animal model expressing HLA for humans is required. In particular, human antigenic peptides useful as cancer vaccines induce specific immune responses when presented to HLA, a human specific mhc class i molecule. Non-human experimental animals lack this HLA and therefore cannot be used for in vivo evaluation of cancer vaccines for direct human therapy. Therefore, HLA-expressing animal models for humans are essential in the evaluation of the efficacy of the cancer vaccines described above.

Disclosure of the invention

It is an object of the present invention to provide WT 1-derived cancer antigen peptides having immunogenicity (activity of inducing CTLs) in vivo, cancer vaccines comprising those peptides, and their use as cancer vaccines, and methods of treatment and prevention of cancer based thereon.

Recently, animal models expressing HLA-A24 antigen for humans have been prepared, which can be used for evaluating in vivo efficacy, and patent applications requiring the invention have been filed (WO 02/47474, International filing date: 6/20/2002, Applicant: Sumitomo pharmaceuticals Co., Ltd.).

This model makes it possible to evaluate the in vivo efficacy of HLA-A24-restricted cancer antigen proteins and cancer antigen peptides, and genes thereof.

The present inventors evaluated natural and altered peptides derived from WT1 and restricted to HLA-A24 using those animal models for humans. That is, our evaluation of peptides having a peptide sequence (binding motif) expected to bind to HLA-A24 antigen, deduced from the WT1 sequence, using BIMAS software (http:// BIMAS. dcrt. nih. gov/molbio/HLA _ bind /) revealed that, among the following natural peptides:

peptide A: arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 8)

Peptide B: arg Val Pro Gly Val Ala Pro Thr Leu (SEQ ID NO: 7)

Peptide C: arg Trp Pro Ser Cys Gln Lys Lys Phe (SEQ ID NO: 9)

Peptide D: gln Tyr Arg Ile His Thr His Gly Val Phe (SEQ ID NO: 10) and

peptide E: ala Tyr Pro Gly Cys Asn Lys Arg Tyr Phe (SEQ ID NO: 11),

only peptide B (SEQ ID NO: 7) was immunogenic in vivo (activity inducing CTLs).

Furthermore, the present inventors prepared the following modified peptides:

peptide F: arg Tyr Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 2)

Peptide G: arg Tyr Pro Gly Val Ala Pro Thr Leu (SEQ ID NO: 3) and

peptide H: arg Tyr Pro Ser Cys Gln Lys Lys Phe (SEQ ID NO: 4),

all of these peptides were altered in which the amino acid at position 2 of the above peptides A to C was changed to tyrosine (Tyr), and their immunogenicity was evaluated in a similar manner. As a result, the present inventors found that the modified peptide G has higher immunogenicity than its original native peptide B. Likewise, the present inventors found that, although the native type peptides a and C are not immunogenic, their modified type peptides F and H have high immunogenicity (activity of inducing CTLs).

In addition, the present inventors also evaluated the immunogenicity of the following natural peptides (peptides K and L) from human WT1, identified in a search by the BIMAS software as having a peptide sequence expected to bind to HLA-a24 antigen, and the following altered peptides thereof in which the amino acid at position 2 was changed to tyrosine (peptides I and J) in a similar manner:

peptide K: ala Leu Leu Pro Ala Val Pro Ser Leu (SEQ ID NO: 51)

Peptide L: asn Gln Met Asn Leu Gly Ala Thr Leu (SEQ ID NO: 52)

Peptide I: ala Tyr Leu Pro Ala Val Pro Ser Leu (SEQ ID NO: 5) and

peptide J: asn Tyr Met Asn Leu Gly Ala Thr Leu (SEQ ID NO: 6). As a result, the present inventors found that, although the natural type: peptides K and L are not immunogenic (inducing activity of CTLs), but their altered forms: peptides I and J are highly immunogenic in vivo (inducing activity of CTLs).

Based on those findings, the present inventors have conducted extensive studies, such as the nucleotide sequence shown in SEQ ID Nos: 2 to 6 and seq id NO: 7 with or without various modifications can be used as cancer vaccines. The present invention has been completed based on the above findings.

Accordingly, the present invention relates to:

(I) a peptide comprising an amino acid sequence selected from any one of the following groups:

Arg Tyr Phe Pro Asn Ala Pro Tyr Leu(SEQ ID NO：2)，

Arg Tyr Pro Gly Val Ala Pro Thr Leu(SEQ ID NO：3)，

Arg Tyr Pro Ser Cys Gln Lys Lys Phe(SEQ ID NO：4)，

ala Tyr Leu Pro Ala Val Pro Ser Leu (SEQ ID NO: 5), and

asn Tyr Met Asn Leu Gly Ala Thr Leu (SEQ ID NO: 6); or a polypeptide consisting of a sequence selected from SEQ ID NOs: 2, 3, 4, 5, and 6; or

A peptide comprising an altered amino acid sequence which is modified at a position selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, and 6, which have activity of inducing CTLs in an HLA-a 24-restricted manner, excluding peptides comprising SEQ id nos: 7 amino acid sequence; preferably, the peptide according to the invention comprises an altered amino acid sequence wherein the amino acid sequence is represented in a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 5, and 6, wherein the leucine at position 9 is substituted with phenylalanine, tryptophan, isoleucine, or methionine; a peptide according to the invention comprising an altered amino acid sequence wherein the amino acid sequence set forth in SEQ ID NO: 4, the phenylalanine at position 9 is substituted with tryptophan, leucine, isoleucine, or methionine; or a peptide according to the invention comprising an altered amino acid sequence wherein the amino acid sequence set forth in SEQ ID NO: 4 by alanine, serine, or alpha-aminobutyric acid (SEQ ID NO: 66, 67, or 68); or a peptide according to the invention consisting of an altered amino acid sequence comprising an amino acid residue sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, and 6.

(II) a polynucleotide encoding a peptide according to the invention, preferably a polynucleotide encoding a peptide selected from the group consisting of SEQ ID NOs: 2 to 6, and 66 to 68, or a pharmaceutically acceptable salt thereof; or an expression vector comprising a polynucleotide according to the invention; or a transformed cell containing an expression vector according to the present invention; or a method of producing a peptide according to the invention, comprising culturing a cell according to the invention under conditions suitable for expression of the peptide;

(III) an antibody that specifically binds to a peptide according to the invention;

(IV) an antigen-presenting cell that presents on its surface a complex between a cancer antigen peptide derived from the peptide according to the present invention and HLA-a24 antigen, preferably an antigen-presenting cell according to the present invention, whose surface presentation is represented by a polypeptide selected from the group consisting of SEQ ID Nos: 2 to 6 and 66 to 68, and HLA-a24 antigen;

(V) a CTL recognizing a complex between a cancer antigen peptide derived from the peptide according to the present invention and HLA-a24 antigen, preferably a CTL according to the present invention, recognizing a complex consisting of a peptide selected from the group consisting of seq id Nos: 2 to 6 and 66 to 68, and HLA-a24 antigen;

(VI) a pharmaceutical composition comprising a peptide according to the invention, a polynucleotide according to the invention, an expression vector according to the invention, a transformed cell according to the invention, an antigen presenting cell according to the invention, or a CTL according to the invention, together with a pharmaceutically acceptable carrier, in particular a cancer vaccine; and the use of the peptide, expression vector, transformed cell, antigen-presenting cell, or CTL described in the present invention for the production of a cancer vaccine, and a method for treating or preventing cancer, which comprises administering a therapeutically or prophylactically effective amount of the peptide, polynucleotide, expression vector, transformed cell, antigen-presenting cell, or CTL described in the present invention to a cancer patient positive for HLA-A24 and positive for WT1 in need of treatment.

Furthermore, the present invention also provides:

(VII) a pharmaceutical composition comprising any one selected from the group consisting of:

a) comprising Arg Val Pro Gly Val Ala Pro Thr Leu (SEQ ID NO: 7) a peptide of the sequence (I) or (II),

b) a polynucleotide encoding the peptide of a) above,

c) an expression vector comprising the polynucleotide of b) above,

d) a cell comprising the expression vector of c) above,

e) an antigen-presenting cell which presents on its surface a complex between a cancer antigen peptide derived from the peptide represented by a) above and HLA-A24 antigen, and

f) CTLs recognizing a complex between a cancer antigen peptide derived from the peptide represented by the above a) and HLA-A24 antigen, together with a pharmaceutically acceptable carrier; in particular a cancer vaccine; and the use of the above-mentioned peptide, polynucleotide, expression vector, transformed cell, antigen-presenting cell or CTL in the production of a vaccine for cancer, a method for treating or preventing cancer, which comprises administering a therapeutically or prophylactically effective amount of the above-mentioned peptide, polynucleotide, expression vector, transformed cell, antigen-presenting cell or CTL to a cancer patient positive for HLA-A24 and positive for WT1 in need of treatment.

Brief description of the drawings

FIG. 1 shows the preparation of chimeric genes (HLA-A2402/K) for use in constructing the present invention^bGene) H-2K^bSchematic representation of the genomic DNA procedure.

FIG. 2 shows HLA-A2402/K, a method for preparing the chimeric gene of the present invention^bSchematic representation of the genetic process.

FIG. 3 is the amino acid sequence of SEQ ID NO: HLA-A2402/K shown in 33^bThe sequence 1 to 1300 of the genomic sequence and SEQ ID NO: HLA-A2402/K shown in 34^bSequence alignment between sequences 1 to 407 of the cDNA sequence.

FIG. 4 is the amino acid sequence of SEQ ID NO: HLA-A2402/K shown in 33^bSequence 1301 to 2600 of the genomic sequence and SEQ ID NO: HLA-A2402/K shown in 34^bSequence alignment between sequences 408 to 1015 of cDNA sequences.

FIG. 5 is the sequence of SEQ ID NO: HLA-A2402/K shown in 33^bSequence 2601 to 3857 of the genomic sequence and SEQ ID NO: HLA-A2402/K shown in 34^bAlignment of sequences between the 1016 to 1119 bit sequences of the cDNA sequence.

FIG. 6 is a graph showing that when an antigenic peptide derived from HER-2/neu (HER 2/neu) is used_780-788) A pattern of specific CTLs induced upon immunization of HLA-A24 expressing transgenic mice of the invention. The vertical axis and horizontal axis represent cytotoxic activity (% specific cytolysis) and the name of each transgenic mouse, respectively. In the figure, "pep +" refers to the results obtained with target cells treated with a peptide pulse (pulsed with a peptide), "pep-" refers to the results obtained with target cells not treated with any peptide pulse.

FIG. 7 is a graph showing the antigen peptide derived from MAGE-3 (MAGE-3) when used_195-203) A pattern of specific CTLs induced upon immunization of HLA-A24 expressing transgenic mice of the invention. In the figure, the vertical axis, horizontal axis, hollow bar, solid bar have the same meaning as the corresponding description of fig. 6.

FIG. 8 shows the antigen peptide derived from CEA (CEA)_652-660) A pattern of specific CTLs induced upon immunization of HLA-A24 expressing transgenic mice of the invention. In the figure, the vertical axis, the horizontal axis,The meaning of the hollow bars and solid bars is the same as the corresponding description of fig. 6.

FIG. 9 shows that when antigen peptide derived from CEA (CEA) is used_268-277) A pattern of specific CTLs induced upon immunization of HLA-A24 expressing transgenic mice of the invention. In the figure, the vertical axis, horizontal axis, hollow bar, solid bar have the same meaning as the corresponding description of fig. 6.

FIG. 10 shows the antigenic peptides (peptide A, WT 1) when used with human WT 1-derived antigen_126-134) The transgenic mice expressing HLA-A24 of the present invention were immunized with a pattern that failed to induce specific CTLs. In the figure, the vertical axis, horizontal axis, hollow bar, solid bar have the same meaning as the corresponding description of fig. 6.

FIG. 11 shows that when an antigenic peptide derived from human WT1 (peptide B, WT 1)_302-310) A pattern of specific CTLs induced upon immunization of HLA-A24 expressing transgenic mice of the invention. In the figure, the vertical axis, horizontal axis, hollow bar, solid bar have the same meaning as the corresponding description of fig. 6.

FIG. 12 shows that when an antigenic peptide derived from human WT1 (peptide C, WT 1)_417-425) When transgenic mice expressing HLA-A24 of the present invention were immunized, no specific CTLs pattern was induced. In the figure, the vertical axis, horizontal axis, hollow bar, solid bar have the same meaning as the corresponding description of fig. 6.

FIG. 13 shows that when an antigenic peptide derived from human WT1 (peptide D, WT 1)_285-294) When transgenic mice expressing HLA-A24 of the present invention were immunized, no specific CTLs pattern was induced. In the figure, the vertical axis, horizontal axis, hollow bar, solid bar have the same meaning as the corresponding description of fig. 6.

FIG. 14 shows that when an antigenic peptide derived from human WT1 (peptide E, WT 1)_326-335) When transgenic mice expressing HLA-A24 of the present invention were immunized, no specific CTLs pattern was induced. In the figure, the vertical axis, horizontal axis, hollow bar, solid bar have the same meaning as the corresponding description of fig. 6.

FIG. 15 is a graph showing that when an altered peptide (peptide F) is used, an antibody derived from human WT1Propeptide (peptide A, WT1_126-134) When the transgenic mice of the present invention expressing HLA-A24 were immunized with tyrosine instead of the amino acid residue at position 2, they induced specific CTLs. In the figure, the vertical axis, horizontal axis, hollow bar, solid bar have the same meaning as the corresponding description of fig. 6.

FIG. 16 is a view showing the use of an altered peptide (peptide G) in which the antigenic peptide derived from human WT1 (peptide B, WT 1)_302-310) When the transgenic mice expressing HLA-A24 of the present invention were immunized with tyrosine instead of the amino acid residue at position 2, a pattern of specific CTLs was induced. In the figure, the vertical axis, horizontal axis, hollow bar, solid bar have the same meaning as the corresponding description of fig. 6.

FIG. 17 is a view showing that an altered peptide (peptide H) in which an antigenic peptide derived from human WT1 (peptide C, WT 1) was used_417-425) When the transgenic mice of the present invention expressing HLA-A24 were immunized with the amino acid residue at position 2 of (1) changed to tyrosine, a pattern of specific CTLs was induced. In the figure, the vertical axis, horizontal axis, hollow bar, solid bar have the same meaning as the corresponding description of fig. 6.

FIG. 18 is a view showing that an altered peptide (peptide I) in which the antigenic peptide derived from human WT1 (peptide K, WT) was used_110-18) When the transgenic mice of the present invention expressing HLA-A24 were immunized with the amino acid residue at position 2 of (1) changed to tyrosine, a pattern of specific CTLs was induced. In the figure, the vertical axis, horizontal axis, hollow bar, solid bar have the same meaning as the corresponding description of fig. 6.

FIG. 19 is a view showing the use of an altered peptide (peptide J) in which the antigenic peptide derived from human WT1 (peptide L, WT 1)_239-247) In (2) to tyrosine, and when the transgenic mice expressing HLA-A24 of the present invention were immunized, specific CTLs were induced. In the figure, the vertical axis, horizontal axis, hollow bar, solid bar have the same meaning as the corresponding description of fig. 6.

Fig. 20 is a graph showing altered peptides relative to native peptides: graph of the results of cross-reaction detection of peptide H induced effector cells. In the figure, the vertical axis represents the CTL induction activity (% specific cytolysis), and the horizontal axis represents the name of each transgenic mouse. Likewise, in the figure, open bars indicate results obtained with target cells pulsed with the altered peptide (peptide H), dotted bars indicate results obtained with target cells pulsed with the native peptide (peptide C), and filled bars indicate results obtained with target cells not pulsed with any peptide.

FIG. 21 shows that when an antigenic peptide derived from human WT1 (peptide K, WT 1)_10-18) When the transgenic mice expressing HLA-A24 of the present invention were immunized, no specific CTL was induced. In the figure, the vertical axis, horizontal axis, hollow bar, solid bar have the same meaning as the corresponding description of fig. 6.

FIG. 22 shows that when an antigenic peptide derived from human WT1 (peptide L, WT 1)_239-347) When the transgenic mice expressing HLA-A24 of the present invention were immunized, no specific CTL was induced. In the figure, the vertical axis, horizontal axis, hollow bar, solid bar have the same meaning as the corresponding description of fig. 6.

FIG. 23 shows that when an antigenic peptide derived from human WT1 (peptide B, WT 1)_302-310) Or an altered peptide thereof (peptide G) obtained by changing the amino acid residue at position 2 in peptide B to tyrosine upon in vitro stimulation of peripheral blood mononuclear cells from a healthy donor positive for HLA-a2402, induces CTLs. In the figure, the vertical axis represents cytotoxic activity, and the horizontal axis represents the ratio of effector cells (E) to target cells (T), E/T. Filled circles and filled triangles represent cytotoxic activity of effector cells stimulated with multiply altered and native peptides, respectively.

FIG. 24 shows that when an antigenic peptide derived from human WT1 (peptide B, WT 1)_302-310) Or an altered peptide thereof (peptide G) obtained by changing the amino acid residue at position 2 in peptide B to tyrosine, induces CTLs when peripheral blood mononuclear cells from a healthy donor positive for HLA-a2402 are stimulated in vitro. In the figure, the vertical axis represents cytotoxic activity, and the horizontal axis represents the ratio of effector cells (E) to target cells (T), E/T. Filled circles and filled triangles represent peptides that were multiply altered on RERF-LC-AI cells and LK87 cells, respectivelyCytotoxic activity of stimulated effector cells. Open circles, open triangles and open squares represent cytotoxic activity of effector cells stimulated with native peptides on RERF-LC-AI cells, LK87 cells and 11-18 cells, respectively.

FIG. 25 is a graph showing that specific CTLs are induced when a transgenic mouse expressing HLA-A24 is immunized with peptide H. In the figure, the vertical axis represents cytotoxic activity (% specific cytolysis), and the horizontal axis represents the E/T ratio. The filled circles and the open circles represent the results obtained with target cells pulsed with peptide H (immunogenic peptide), and with cells not pulsed with any peptide, respectively.

FIG. 26 is a graph showing that specific CTLs are induced when a transgenic mouse expressing HLA-A24 is immunized with peptide M. In the figure, the vertical axis, horizontal axis, filled circles and open circles have the same meaning as described in connection with fig. 25.

FIG. 27 is a graph showing that specific CTLs are induced when transgenic mice expressing HLA-A24 are immunized with peptide N. In the figure, the vertical axis, horizontal axis, filled circles and open circles have the same meaning as described in connection with fig. 25.

FIG. 28 is a graph showing that specific CTLs are induced when transgenic mice expressing HLA-A24 are immunized with peptide O. In the figure, the vertical axis, horizontal axis, filled circles and open circles have the same meaning as described in connection with fig. 25.

Fig. 29 shows the peptide corresponding to the unsubstituted peptide: substituted peptide of peptide H: peptide M, results of cross-reaction experiments with induced effector cells. In the figure, the vertical axis represents CTL-inducing activity (% specific cytolysis), and the horizontal axis represents the E/T ratio. Likewise, in the figure, filled circles, filled squares and open circles indicate the results obtained with target cells pulsed with peptide M (immunogenic peptide), peptide H and with cells not pulsed with any peptide.

FIG. 30 is a graph showing the following substitution with the corresponding unsubstituted peptide: substituted peptide of peptide H: peptide N, results of cross-reaction experiments with induced effector cells. In the drawings, the vertical axis, the horizontal axis, the solid circle, the solid square, and the hollow circle have the same meanings as in the corresponding description of fig. 29.

Best Mode for Carrying Out The Invention

(I) The peptides according to the invention

The peptide of the present invention is derived from human WT1(cell., 60: 509, 1990, NCBI database Access No. XP _034418, SEQ ID NO: 1) and has an activity of inducing CTLs in vivo in a HLA-A24-restricted manner (immunogenicity).

The peptides of the present invention have the property of being presented on antigen-presenting cells to induce CTLs in vivo in an HLA-A24 antigen-restricted manner. This property can be tested using animal models for HLA-A24 described in detail in the reference protocols below.

The peptides of the invention include a peptide selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, and 6, as long as the cancer antigen peptide derived from the peptide can be presented on antigen-presenting cells to induce CTLs. Typical lengths of the peptide amino acid residues are usually 9 to 100, preferably 9 to 50, more preferably 9 to 30, more preferably 9 to 20, even more preferably 9 to 11. In the context of the present invention, a cancer antigen peptide is defined as a peptide that is capable of causing CTL inducing activity when presented to antigen presenting cells.

The peptides of the present invention can be prepared according to methods generally used in peptide chemistry. Examples of these preparation processes are those described in the following documents: including "PeptideSeynthesis", Interscience, New York, 1966; "The Proteins", vol.2, Academic Press Inc., New York, 1976; "Pepuchido-Gosei", Maruzen co.ltd., 1975; "Pepuchido-Gosei-no-Kiso-to-Jikkenn", Maruzen Co. Ltd., 1985; and "Iyakuhin-no-Kaihatu, Zoku, vol.14, Peputido-Gosei", Hirokawa Shoten, 1991.

The peptide of the present invention can also be prepared according to conventional DNA synthesis and genetic engineering methods based on sequence information of a polynucleotide encoding the peptide of the present invention. For example, DNA synthesis, construction of various plasmids, transfection of the above plasmids into host cells, culture of transformants, and recovery of proteins from the culture can be carried out according to methods known to those skilled in the art, methods described in the literature (molecular cloning, T. Maniatis et al, CSH laboratories (1983), DNA cloning, DM. glover, IRL PRESS (1985)), or methods described in (II) below.

Specific examples of the peptides according to the invention are provided below

(1) Comprises a sequence selected from SEQ ID NO: 2 to 6 in the sequence of any amino acid sequence

As mentioned above, the present invention is based on the amino acid sequence as shown in SEQ ID NO: 2 to 6 the altered peptides derived from WT1 have a novel finding of inducing CTLs activity in vivo. Previously it has not been known as SEQ ID NO: the novel peptides shown in FIGS. 2 to 6 do have the activity of inducing CTLs in vivo. Peptides comprising any of those altered peptides can be used as active ingredients contained in compositions for inducing CTLs or in cancer vaccines for use in cancer immunotherapy.

Specifically, the peptide of the present invention comprises any one of the following sequences:

Arg Tyr Phe Pro Asn Ala Pro Tyr Leu(SEQ ID NO：2)，

Arg Tyr Pro Gly Val Ala Pro Thr Leu(SEQ ID NO：3)，

Arg Tyr Pro Ser Cys Gln Lys Lys Phe(SEQ ID NO：4)，

ala Tyr Leu Pro Ala Val Pro Ser Leu (SEQ ID NO: 5), and

Asn Tyr Met Asn Leu Gly Ala Thr Leu(SEQ ID NO：6)。

among them, a peptide comprising the sequence of Arg Tyr Pro Ser Cys Gln Lys Lys Phe (SEQ ID NO: 4) and a peptide comprising the sequence of Ala Tyr Leu Pro Ala Val Pro Ser Leu (SEQ ID NO: 5) are preferable.

More specific examples of the peptide according to the present invention include peptides as shown in the following (1-1) to (1-4).

(1-1) a polypeptide consisting of a sequence selected from the group consisting of SEQ ID NOs: 2 to 6 in the sequence of any amino acid

Consists of SEQ ID NOs: specific examples of the peptide consisting of any one of amino acid sequences 2 to 6 include the following cancer antigen peptides:

a cancer antigen peptide consisting of the sequence of Arg Tyr Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 2),

a cancer antigen peptide consisting of the sequence of Arg Tyr Pro Gly Val Ala Pro Thr Leu (SEQ ID NO: 3),

a cancer antigen peptide consisting of the sequence of Arg Tyr Pro Ser Cys Gln Lys Lys Phe (SEQ ID NO: 4),

a cancer antigen peptide consisting of the sequence Ala Tyr Leu Pro Ala Val Pro Ser Leu (SEQ ID NO: 5), and

a cancer antigen peptide consisting of the sequence Asn Tyr Met Asn Leu Gly Ala Thr Leu (SEQ ID NO: 6).

Among them, preferred are a cancer antigen peptide consisting of the sequence Arg Tyr Pro Ser Cys Gln Lys Lys Phe (SEQ ID NO: 4) and a cancer antigen peptide consisting of the sequence Ala Tyr Leu Pro Ala Val ProSer Leu (SEQ ID NO: 5). These peptides can be prepared according to the conventional methods for peptide synthesis described above. The activity of these peptides in inducing CTLs in vivo can be tested using the animal models for humans described below.

(1-2) comprises SEQ ID NOs: 2 to 6, and a motif-structure-containing peptide

It is known that there are many subtypes in HLA molecules, and the amino acid sequence of an antigen peptide binding to each subtype follows a certain rule (binding motif). It is also known that, for the binding motif of HLA-A24, the amino acid at position 2 is tyrosine (Tyr), phenylalanine (Phe), methionine (Met), or tryptophan (Trp), and in a peptide consisting of 8 to 11 amino acid residues, the amino acid at the C-terminus is phenylalanine (Phe), leucine (Leu), isoleucine (Ile), tryptophan (Trp), or methionine (Met) (J.Immunol., 152, p3913, 1994, Immunogenetics, 41, p178, 1995, J.Immunol., 155, p.4307, 1994).

Based on this rule, examples of the peptide according to the present invention also include a peptide consisting of 10 amino acid residues in which Phe, Leu, Ile, Trp, or Met is added to the C-terminus of any one of cancer antigen peptides consisting of 9 amino acid residues:

Arg Tyr Phe Pro Asn Ala Pro Tyr Leu(SEQ ID NO：2)，

Arg Tyr Pro Gly Val Ala Pro Thr Leu(SEQ ID NO：3)，

Arg Tyr Pro Ser Cys Gln Lys Lys Phe(SEQ ID NO：4)，

ala Tyr Leu Pro Ala Val Pro Ser Leu (SEQ ID NO: 5), or

Asn Tyr Met Asn Leu Gly Ala Thr Leu (SEQ ID NO: 6), and a peptide consisting of 11 amino acid residues in which Phe, Leu, Ile, Trp, or Met is further added to the C-terminus of any of the peptides consisting of 10 amino acid residues, all of which have an activity of inducing CTLs in vivo. These peptides can be synthesized according to the conventional methods for peptide synthesis described above. The activity of these peptides in inducing CTLs in vivo can be tested using the animal models for humans described below.

(1-3) comprises SEQ ID NOs: epitope peptide of any one of amino acid sequences 2 to 6

Recently, a number of peptides (epitope peptides) in which CTL epitopes (antigen peptides) are linked to each other have been demonstrated to have an activity of efficiently inducing CTLs in vivo. For example, Journal of Immunology 1998, 161: 3186-3194 it is described that peptides of about 30 amino acids in which HLA-A2, -A3, -A11, B53-restricted CTL epitopes derived from cancer antigen protein, PSA, are linked to each other induce CTLs specific to the relevant CTL epitopes in vivo.

Likewise, it has been demonstrated that a peptide (epitope peptide) in which a CTL epitope and a helper epitope are linked to each other can effectively induce CTLs. Helper epitopes refer to peptides that activate CD4 positive T cells (Immunity., 1: 751, 1994), and are known to include HBVc128-140 derived from hepatitis B virus and TT947-967 derived from tetanus toxin. It is believed that CD 4-positive T cells activated by the helper epitope play an important role in immune responses to cancer eradication, because they play roles in, for example, inducing CTL differentiation and maintaining CTLs, and activating effector cells including macrophages. Examples of such peptides in which a helper epitope and a CTL epitope are linked to each other, for example, Journal of Immunology1999, 162: 3915-3925 describes a CTLs effective in inducing a response to the relevant epitope in vivo by DNA encoding peptides linked to 6 HLA-A2-restricted antigen peptides, 3 HLA-A11-restricted antigen peptides derived from HBV, and a helper epitope (minigene). In addition, a peptide in which a CTL epitope (a Cancer antigen peptide consisting of amino acid residues 280 to 288 of melanoma antigen, gp100) and a helper epitope (tetanus toxin-derived T helper epitope) are linked to each other has been examined in Clinical experiments (Clinical Cancer Res., 2001, 7: 3012-3024).

Based on these findings, a peptide (epitope peptide) in which a cancer antigen peptide or a peptide shown in the above (1-1) or (1-2) is linked to various epitopes and has an activity of inducing CTLs in vivo is also exemplified as the peptide according to the present invention.

In case the epitope linked to the cancer antigen peptide of the present invention is a CTL epitope, usable CTL epitopes include those derived from WT1, which are restricted by HLA-A1, -A0201, -A0204, -A0205, -A0206, -A0207, -A11, -A24, -A31, -A6801, -B7, -B8, -B2705, -B37, -Cw0401, -Cw0602, and the like. Two or more epitopes may be linked, and a CTL epitope may be 8 to 14 amino acid residues in length based on analysis of antigen peptides binding to various HLA molecules (Immunogenetics, 41: 178, 1995).

If the epitope to which the antigenic peptide of the present invention is linked is a helper epitope, the helper epitope may be, for example, HBVc128-140 derived from hepatitis B virus and TT947-967 derived from tetanus toxin as described above. The length of the helper epitope can be about 13 to about 30, preferably about 13 to about 17 amino acid residues.

Specific examples of epitope skins as described herein include SEQ ID NOs: 2 to 6, to a helper epitope. More specifically, for example, SEQ ID NOs: 2 to 6 to a tetanus toxin-derived helper epitope (e.g. Phe Asn Asn Phe Thr Val Ser PheTrp Leu Arg Val Pro Lys Val Ser Ala Ser His Leu Glu; SEQ ID no: 32), and SEQ ID NOs: 2 to 6 to Ala Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile GlyIle Thr Glu Leu (SEQ ID NO: 50, Clinical Cancer Res., 2001, 7: 3012-3024).

Those peptides in which various epitopes are linked to each other can be prepared according to the usual methods for peptide synthesis described above. These peptides can also be prepared according to conventional DNA synthesis and genetic engineering methods based on sequence information of polynucleotides encoding peptides formed by linking various epitopes to each other. That is, these peptides can be prepared by inserting polynucleotides into a known expression vector, transforming host cells with a recombinant expression vector, culturing the transformant, and recovering epitope peptides in which various desired epitopes are linked to each other from the culture. This can be carried out according to the methods described in the literature (molecular cloning, T.Maniatis et al, CSH laboratories (1983), DNA cloning, DM.Glover, IRL PRESS (1985)), or those described in (II) below.

The activity of inducing CTLs in vivo of epitope peptides prepared by linking various epitopes to each other can be examined using an animal model for humans described in the reference scheme below.

(1-4) comprises SEQ ID NOs: 2 to 6, and wherein the amino group of the N-terminal amino acid or the carboxyl group of the C-terminal amino acid is modified

The amino group of the N-terminal amino acid or the carboxyl group of the C-terminal amino acid in the peptide of the present invention as described in the above (1-1) to (1-3) may be modified.

In the context of the present invention, the modifying group of the amino group of the N-terminal amino acid includes an alkyl group having 1 to 6 carbon atoms, a phenyl group, a cycloalkyl group, an acyl group, etc., of which 1 to 3 groups may be selected. Examples of the acyl group include an alkanoyl group having 1 to 6 carbon atoms, an alkanoyl group having 1 to 6 carbon atoms substituted with a phenyl group, a carbonyl group substituted with a cycloalkyl group having 5 to 7 carbon atoms, an alkylsulfonyl group having 1 to 6 carbon atoms, a benzenesulfonyl group, an alkoxycarbonyl group having 2 to 6 carbon atoms, an alkoxycarbonyl group substituted with a phenyl group, a carbonyl group substituted with a cycloalkoxy group having 5 to 7 carbon atoms, a phenoxycarbonyl group, and the like.

Peptides in which the carboxyl group of the C-terminal amino acid is modified include esters and amides. Specific esters include C1-C6 alkyl esters, C0-C6 alkyl esters substituted with phenyl, C5-C7 cycloalkyl esters, and the like, while specific amides include amides, amides substituted with one or two C1-C6 alkyl groups, amides substituted with one or two C0-C6 alkyl groups substituted with phenyl, amides forming azacycloalkanes containing an amide nitrogen atom numbered 5 to 7, and the like.

(2) A peptide comprising an altered amino acid sequence, wherein the amino acid sequence is represented in a sequence selected from the group consisting of SEQ ID NOs: 2 to 6, which contains an altered amino acid residue (multiple altered peptide)

As mentioned above, the present invention is based on the nucleic acid sequences as shown in SEQ ID NOs derived from WT 1: the altered peptides shown in 2 to 6 have a novel finding of inducing CTLs activity in vivo. Additional changes to the amino acids of those peptides that have the activity of inducing CTLs in vivo may result in the production of multiple altered peptides that have the same or more potent activity of inducing CTLs. In this respect, the present invention provides a polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 2 to 6 (those peptides may be referred to as multiply-altered peptides hereinafter).

In particular, the invention provides peptides comprising an altered amino acid sequence, wherein the amino acid sequence is encoded in a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5 and 6, which has an activity of inducing CTLs, provided that a peptide comprising the amino acid sequence of SEQ ID NO: 7 amino acid sequence.

Herein, "alteration" of an amino acid residue means substitution, deletion and/or addition of one or several amino acid residues, preferably substitution of an amino acid residue. For changes involving substitution of amino acid residues, the number and position of amino acid residues to be substituted can be arbitrarily determined as long as the activity of inducing CTLs in vivo is retained. Examples of such peptides comprising an altered amino acid sequence include those shown below.

As described above, it is known that, for the binding motif of HLA, the amino acid at position 2 is tyrosine (Tyr), phenylalanine (Phe), methionine (Met), or tryptophan (Trp), and the amino acid at the C-terminal end of a peptide consisting of 8 to 11 amino acid residues is phenylalanine (Phe), leucine (Leu), isoleucine (Ile), tryptophan (Trp), or methionine (Met) (j.immunol., 152, p3913, 1994, Immunogenetics, 41, p178, 1995, j.immunol., 155, p4307, 1994). Based on this rule, the multiply altered peptides according to the invention may comprise substitutions of amino acid residues in the above motifs for peptides represented in SEQ ID NOs: 2 to 6, and/or at position 2 and/or 9 in the amino acid sequence.

Specific examples of the multiple altered peptide comprising the altered amino acid residue at position 2 include peptides comprising any one of the following amino acid sequences and having an activity of inducing CTLs in vivo:

Arg Phe Phe Pro Asn Ala Pro Tyr Leu(SEQ ID NO：53)，

Arg Trp Phe Pro Asn Ala Pro Tyr Leu(SEQ ID NO：54)，

Arg Phe Pro Gly Val Ala Pro Thr Leu(SEQ ID NO：55)，

Arg Met Pro Gly Val Ala Pro Thr Leu(SEQ ID NO：56)，

Arg Trp Pro Gly Val Ala Pro Thr Leu(SEQ ID NO：57)，

Arg Phe Pro Ser Cys Gln Lys Lys Phe(SEQ ID NO：58)，

Arg Met Pro Ser Cys Gln Lys Lys Phe(SEQ ID NO：59)，

Ala Phe Leu Pro Ala Val Pro Ser Leu(SEQ ID NO：60)，

Ala Met Leu Pro Ala Val Pro Ser Leu(SEQ ID NO：61)，

Ala Trp Leu Pro Ala Val Pro Ser Leu(SEQ ID NO：62)，

Asn Phe Met Asn Leu Gly Al Thr Leu(SEQ ID NO：63)，

asn Met Met Asn Leu Gly Ala Thr Leu (SEQ ID NO: 64), and

Asn Trp Met Asn Leu Gly Ala Thr Leu(SEQ ID NO：65)。

those peptides include those represented by SEQ ID NOs: 53 to 65, which has an activity of inducing CTLs in vivo.

All shown in SEQ ID NOs: 2 to 6 are obtained by modifying the amino acid residue at position 2 in a natural peptide derived from human WT1 to obtain a modified peptide having an activity of inducing CTLs efficiently. In the context of the present invention, the amino acid residue at position 2 of the multiply altered peptide of the invention is preferably tyrosine. On the other hand, the amino acid residue at the C-terminal end of the multiply altered peptide may be altered to an amino acid residue available for the above motif.

Specific examples of the peptides that are multiply altered according to the embodiments of the present invention include peptides comprising any one of the following amino acid sequences, which have an activity of inducing CTLs in vivo:

Arg Tyr Phe Pro Asn Ala Pro Tyr Phe(SEQ ID NO：12)，

Arg Tyr Phe Pro Asn Ala Pro Tyr Trp(SEQ ID NO：13)，

Arg Tyr Phe Pro Asn Ala Pro Tyr Ile(SEQ ID NO：14)，

Arg Tyr Phe Pro Asn Ala Pro Tyr Met(SEQ ID NO：15)，

Arg Tyr Pro Gly Val Ala Pro Thr Phe(SEQ ID NO：16)，

Arg Tyr Pro Gly Val Ala Pro Thr Trp(SEQ ID NO：17)，

Arg Tyr Pro Gly Val Ala Pro Thr Ile(SEQ ID NO：18)，

Arg Tyr Pro Gly Val Ala Pro Thr Met(SEQ ID NO：19)，

Arg Tyr Pro Ser Cys Gln Lys Lys Trp(SEQ ID NO：20)，

Arg Tyr Pro Ser Cys Gln Lys Lys Leu(SEQ ID NO：21)，

Arg Tyr Pro Ser Cys Gln Lys Lys Ile(SEQ ID NO：22)，

Arg Tyr Pro Ser Cys Gln Lys Lys Met(SEQ ID NO：23)，

Ala Tyr Leu Pro Ala Val Pro Ser Phe(SEQ ID NO：24)，

Ala Tyr Leu Pro Ala Val Pro Ser Trp(SEQ ID NO：25)，

Ala Tyr Leu Pro Ala Val Pro Ser Ile(SEQ ID NO：26)，

Ala Tyr Leu Pro Ala Val Pro Ser Met(SEQ ID NO：27)，

Asn Tyr Met Asn Leu Gly Ala Thr Phe(SEQ ID NO：28)，

Asn Tyr Met Asn Leu Gly Ala Thr Trp(SEQ ID NO：29)，

asn Tyr Met Asn Leu Gly Ala Thr Ile (SEQ ID NO: 30), and

Asn Tyr Met Asn Leu Gly Ala Thr Met(SEQ ID NO：31)。

those peptides include those represented by SEQ ID NOs: 12 to 31, which has an activity of inducing CTLs in vivo.

Another embodiment of the present invention includes a cancer antigen peptide containing not only the change of the amino acid residue at position 2 of a peptide comprising multiple changes of the amino acid residue at position 2 but also the above-mentioned change of the C-terminal amino acid residue.

SEQ ID NO: the 4 amino acid sequence contains cysteine residues which can be oxidized to form disulfide bonds in solution. To avoid this, the cysteine residue may be replaced with another amino acid residue such as an alanine residue, a serine residue, or the like, or α -aminobutyric acid which is similar in chemical structure to the cysteine residue, to provide a peptide which is multiply changed.

Specific examples of the peptides that are multiply altered according to the embodiments of the present invention include peptides having an amino acid sequence of any one of the following, which have an activity of inducing CTLs in vivo:

Arg Tyr Pro Ser Ser Gln Lys Lys Phe(SEQ ID NO：66)，

arg Tyr Pro Ser Ala Gln Lys Lys Phe (SEQ ID NO: 67), and

Arg Tyr Pro Ser Abu Gln Lys Lys Phe(SEQ ID NO：68)，

wherein Abu is alpha-aminobutyric acid.

These peptides include those represented by SEQ ID NOs: 66 to 68, which has an activity of inducing CTLs in vivo.

Those peptides can be prepared according to the general methods of peptide synthesis described above. The activity of those peptides to induce CTLs in vivo can be tested using animal models for humans as described in the reference protocol below.

The above-mentioned multiply altered peptide of the present invention may also be modified by maintaining the motif structure shown in the above-mentioned (1-2), linking to a plurality of epitopes shown in the above-mentioned (1-3), or modifying the amino or carboxyl group shown in the above-mentioned (1-4).

The peptides of the present invention are useful, for example, as active ingredients contained in compositions for inducing CTLs or cancer vaccines, or for preparing antigen-presenting cells as described below.

(II) the polynucleotide, expression vector, and transformant of the present invention

The present invention also provides polynucleotides encoding the above-described polypeptides of the invention. The polynucleotide encoding the peptide of the present invention may be in the form of DNA or RNA. Those polynucleotides can be easily prepared based on the amino acid sequence information of the peptide of the present invention and the information of DNAs encoding the peptide of the present invention. Specifically, they can be prepared according to a conventional method for DNA synthesis or PCR amplification.

Examples of polynucleotides of the invention include: polynucleotides encoding a peptide having the sequence Arg Tyr Phe Pro Asn AlaPro Tyr Leu (SEQ ID NO: 2), polynucleotides encoding a peptide having the sequence Arg TyrPro Gly Val Ala Pro Thr Leu (SEQ ID NO: 3), polynucleotides encoding a peptide having the sequence Arg Tyr Pro Ser Cys Gln Lys Lys Phe (SEQ ID NO: 4), polynucleotides encoding a peptide having the sequence Ala Tyr Leu Pro Ala Val Pro SerLeu (SEQ ID NO: 5), polynucleotides encoding a peptide having the sequence Asn Tyr Met AsnLeu Gly Ala Thr Leu (SEQ ID NO: 6), polynucleotides encoding a peptide having the sequence Arg Tyr Pro Ser Ser Gln Lys Lys Phe (SEQ ID NO: 66), polynucleotides encoding a peptide having the sequence Arg Tyr Pro Ser Ala Gln Lys Lys Phe (SEQ ID NO: 67), a polynucleotide encoding a peptide comprising the sequence Arg Tyr Pro SerAbu Gln Lys Lys Phe (SEQ ID NO: 68), wherein Abu is alpha-aminobutyric acid.

Specific examples of the polynucleotide include a polynucleotide encoding a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 2 to 6 and 66 to 68. More specifically, for example, a nucleic acid encoding the amino acid sequence of SEQ ID NO: 2 to 6 and 66 to 68, comprising a polynucleotide encoding a peptide of any one or more of the amino acid sequences of SEQ ID NOs: 2 to 6 and 66 to 68 to a tetanus toxin-derived helper epitope (e.g., Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg ValPro Lys Val Ser Ala Ser His Leu Glu; SEQ ID NO: 32), and SEQ ID NO: 2 to 6 and 66 to 68 to Ala Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr GluLeu (SEQ ID NO: 50, Clinical Cancer Res., 2001, 7: 3012-3024).

The polynucleotide of the present invention thus prepared may be inserted into an expression vector to prepare a recombinant expression vector for expressing the peptide of the present invention.

The expression vector used herein may be appropriately selected depending on the host and the purpose of use, and includes plasmids, phage vectors, and viral vectors.

Examples of vectors for use in an E.coli host include plasmids such as pUC118, pUC119, pBR322, and pCR3, and phage vectors such as λ ZAPII, and λ gt 11. Examples of vectors for yeast cells include pYES2, and pYEUra 3. Mutexamples of vectors for insect cell hosts include pAcSGHisNT-A. Examples of vectors for use in animal cell hosts include plasmids such as pKCR, pCDM8, pGL2, pcDNA3.1, pRc/RSV, and pRc/CMV, and viral vectors such as retroviral vectors, adenoviral vectors, and adeno-associated viral vectors.

If desired, those vectors may contain such factors as promoters for inducible expression, genes encoding signal sequences, selectable marker genes, terminators, and the like.

Likewise, the vector may contain an additional sequence of thioredoxin, histidine tag, or GST (glutathione S-transferase) forming a fusion protein to facilitate isolation and purification. In this example, GST fusion protein expression vectors (i.e., pGEX4T) containing promoters suitable for operation in the host cell (lac, tac, trc, trp, CMV, SV40 early promoter, etc.), vectors containing tag sequences such as Myc, His (i.e., pcdna3.1/Myc-His), and vectors expressing fusion proteins containing thioredoxin or His tag (pET32a) can be used.

The activity of inducing CTLs in vivo by the polynucleotide or the expression vector containing the polynucleotide can be examined in an animal model for humans as described in the reference scheme below.

The polynucleotide of the present invention or an expression vector containing the polynucleotide can be used, for example, for preparing the peptide of the present invention, gene therapy described below, or preparing antigen presenting cells described below.

The thus-prepared expression vector of the present invention may be transformed into a host to prepare a transformant containing the expression vector.

Host cells for use herein include E.coli, yeast, insect cells, and animal cells. Coli includes E.coli K-12 lines, such as HB101 strain, C600 strain, JM109 strain, DH5 alpha strain and AD494(DE3) strain. The yeast includes Saccharomyces cerevisiae. Animal cells include L929 cells, BALB/C3T3 cells, C127 cells, CHO cells, COS cells, Vero cells, and Hela cells. The insect cell includes sf 9.

The various expression vectors may be used to transform the host cells using conventional transformation methods appropriate for the respective host cells. Specific methods include a calcium phosphate method, a DEAE-dextran method, electroporation, and a method using a lipid for gene transfer (Lipofectamine, Lipofectin, Gibco-BRL). After transformation, the transformant is cultured in a conventional medium containing a selection marker, thereby selecting a transformant in which the above-mentioned expression vector has been transferred into the host cell.

The thus-prepared transformant can be cultured under appropriate conditions to prepare the peptide of the present invention. The polypeptide may be further isolated and purified according to conventional methods of biochemical purification. Examples of the purification method include salt precipitation, ion exchange chromatography, adsorption chromatography, affinity chromatography, and gel filtration chromatography. When the polypeptide of the present invention is expressed as a fusion protein containing thioredoxin, a histidine tag, GST, etc., the polypeptide can be isolated and purified by a purification method based on the characteristics of such a fusion protein or tag.

(III) antibodies of the invention

The invention provides antibodies that specifically bind to a peptide according to the invention. The antibody of the present invention is not limited to a specific antibody, and may be a polyclonal antibody or a monoclonal antibody against the peptide of the present invention as an immunogen.

As described above, the antibodies of the present invention are not limited to specific antibodies as long as they can specifically bind to the peptides of the present invention, and specific examples thereof include antibodies specifically binding to a peptide consisting of a sequence selected from the group consisting of SEQ ID NOs: 2 to 6 and 66 to 68, respectively.

Methods for preparing Antibodies are well known and the Antibodies of the invention can be prepared according to methods generally known in the art (Current protocols in Molecular biology. Ausubel et al, (1987) Publish. John Wiley and sons. section11.12 to 11.13, Antibodies; A Laboratory Manual, Lane, H, D. et al, Cold Spring Harber Laboratory Press publication, New York 1989).

Specifically, the antibody can be produced by immunizing a non-human animal such as a rabbit with a peptide of the present invention (for example, a cancer antigen peptide consisting of any one amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 6 and 66 to 68) as an immunogen, and then obtaining the antibody from the serum of the immunized animal in a conventional manner. On the other hand, a monoclonal antibody can be prepared by a method of immunizing a non-human animal such as a mouse with a peptide of the present invention (for example, a cancer antigen peptide consisting of any one amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 6 and 66 to 68), preparing a hybridoma from the obtained spleen cell and myeloma cell by cell fusion, and then obtaining an antibody from the hybridoma (Current protocols Molecular Biology, Ausubel et al (1987) Publish.John Wileyand sons. section 11.4 to 11.11).

Antibodies against the peptides of the invention can be prepared in a manner that enhances the immune response using a variety of adjuvants appropriate to the host. Examples of adjuvants include Freund's adjuvant, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin and polyalcohols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and adjuvants for humans such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum (Corynebacterium-parvum).

As described above, an antibody recognizing a peptide, and an antibody neutralizing the activity of the peptide can be easily prepared by appropriately immunizing an animal with the peptide of the present invention in a conventional manner. Such antibodies can be used in methods such as affinity chromatography, immunodiagnosis, and the like. The immunodiagnostic method may be selected from among methods such as immunoblotting, Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), fluorescence or luminescence assay. Immunodiagnosis can be used to diagnose cancers expressing WT1 gene, such as gastric cancer, colon cancer, lung cancer, breast cancer, embryonal cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, and ovarian cancer.

(IV) the antigen-presenting cell of the present invention

The present invention provides antigen-presenting cells that present on their surface a complex formed between a cancer antigen peptide derived from the peptide of the present invention and HLA-A24 antigen.

The examples described below demonstrate that administration of the peptide of the present invention can induce CTLs, which indicates that there are antigen-presenting cells capable of presenting on their surface a complex formed between a cancer antigen peptide derived from the peptide of the present invention and HLA-A24 antigen in peripheral blood mononuclear cells, followed by induction of CTLs capable of specifically destroying cancer cells whose surfaces present such a complex. Those antigen-presenting cells which present on their surface a complex formed between a cancer antigen peptide derived from the peptide of the present invention and HLA-A24 antigen can be used in the cell therapy (DC therapy) described below.

The antigen-presenting cells of the present invention are not limited to specific cells as long as they are capable of presenting on their surface a complex formed between a cancer antigen peptide derived from the peptide of the present invention and HLA-a24 antigen, and preferably include cells whose surface presentation is represented by SEQ ID NOs: 2 to 6 and 66 to 68, and HLA-a 24.

Antigen-presenting cells for use in cell therapy can be prepared by, for example, isolating antigen-presenting cells from a cancer patient and pulsing the cells with the peptide of the present invention in vitro, or transforming the cells with the polynucleotide of the present invention or an expression vector containing the polynucleotide, to present a complex formed between the HLA-a24 antigen and a cancer antigen peptide derived from the peptide of the present invention. In the context of the present invention, the "cell having antigen-presenting ability" is not limited to a specific cell as long as it is a cell that expresses on its cell surface the HLA-A24 antigen allowing presentation of the peptide of the present invention, and is preferably a dendritic cell that is believed to have particularly high antigen-presenting ability, for example.

The substance for pulsing to the antigen-presenting cells may be the peptide of the present invention, as well as a polynucleotide encoding the peptide of the present invention and an expression vector containing the polynucleotide.

The antigen-presenting cells of the present invention can be prepared, for example, by isolating antigen-presenting cells from a Cancer patient, pulsing the cells in vitro with a peptide of the present invention (e.g., a Cancer antigen peptide consisting of an amino acid sequence of any one of SEQ ID NOs: 2 to 6 and 66 to 68), and preparing a complex between HLA-A24 antigen and a Cancer antigen peptide derived from the peptide of the present invention (Cancer Immunol. Immunother., 46: 82, 1998, J.Immunol., 158: p1796, 1997, Cancer Res., 59: p1184, 1999). When dendritic cells are used, the antigen-presenting cells of the present invention can be prepared, for example, by isolating lymphocytes from peripheral blood of a cancer patient using the Ficoll method, removing non-adherent cells, culturing adherent cells in the presence of GM-CSF and IL-4 to induce dendritic cells, and culturing and pulsing the dendritic cells using the peptides of the present invention.

When the antigen-presenting cell of the present invention is prepared by transforming the above-mentioned cell having antigen-presenting ability with a polynucleotide encoding a peptide of the present invention (for example, a polynucleotide encoding a peptide having a sequence of any one of SEQ ID NOs: 2 to 6 and 66 to 68), or an expression vector containing the polynucleotide, such a polynucleotide prepared in the form of DNA can be referred to, for example, as a cancer Res., 56: p5672, 1996, or j.immunol., 161: p5607, 1998. Similarly, such polynucleotides prepared in RNA form also allow for the preparation of antigen presenting cells, as can be seen, for example, in j.exp.med., 184: p465, 1996.

The thus-prepared antigen-presenting cells of the present invention can be used as an active ingredient contained in a composition for inducing CTLs or a cancer vaccine, or used for the following cell therapy (DC therapy).

(V) CTLs of the present invention

The present invention provides CTLs recognizing a complex formed between a cancer antigen peptide derived from the peptide of the present invention and HLA-A24 antigen

The examples described below demonstrate that administration of the peptide of the present invention can induce CTLs, which indicates that there are antigen-presenting cells capable of presenting on their surface a complex formed between a cancer antigen peptide derived from the peptide of the present invention and HLA-A24 antigen in peripheral blood mononuclear cells, followed by induction of CTLs capable of specifically destroying cancer cells whose surfaces present such a complex. CTLs that specifically recognize a complex formed between a cancer antigen peptide derived from the peptide of the present invention and HLA-A24 antigen can be used for immunotherapy as described below.

The CTLs of the present invention are not limited to specific CTLs as long as they specifically recognize a complex formed between a cancer antigen peptide derived from the peptide of the present invention and HLA-a24 antigen, and particularly include a peptide specifically recognizing a peptide consisting of SEQ ID NOs: 2 to 6 and 66 to 68, and a complex formed between a cancer antigen peptide consisting of an amino acid sequence of any one of 2 to 6 and HLA-a24 antigen.

For the preparation of CTLs for use in immunotherapy, for example, peripheral lymphocytes are isolated from a patient, and in vitro stimulation is carried out using a peptide of the present invention (e.g., a cancer antigen peptide consisting of an amino acid sequence of any one of SEQ ID NOs: 2 to 6 and 66 to 68), or a polynucleotide encoding a peptide of the present invention (e.g., a polynucleotide encoding a peptide containing an amino acid sequence of any one of SEQ ID NOs: 2 to 6 and 66 to 68) or an expression vector containing the polynucleotide (Journal of Experimental Medicine 1999, 190: 1669).

The CTLs of the present invention thus prepared can be used as active ingredients contained in cancer vaccines or in immunotherapy employed.

(VI) pharmaceutical compositions, uses, and methods as cancer vaccines

The peptides of the present invention, the polynucleotides of the present invention, the expression vectors of the present invention, the antigen-presenting cells of the present invention, and the CTLs of the present invention can be used in a suitable form as an active ingredient contained in a composition for inducing CTLs or a cancer vaccine, and the following examples are given.

(6-1) cancer vaccine comprising the peptide of the present invention as an active ingredient

CTLs induced by the peptides of the present invention having CTLs-inducing activity can destroy cancer cells by their cytotoxic activity and lymphokine products. Therefore, the peptide of the present invention can be used as an active ingredient in a cancer vaccine for treating or preventing cancer. In a specific embodiment, the present invention provides a cancer vaccine (pharmaceutical composition useful as a cancer vaccine) comprising the peptide of the present invention as an effective ingredient. When the cancer vaccine of the present invention is administered to HLA-A24-positive and WT 1-positive cancer patients, peptides (e.g., cancer antigen peptides consisting of any one of the amino acid sequences of SEQ ID NOs: 2 to 6 and 66 to 68) are presented on the HLA-A24 antigen of antigen-presenting cells, and then CTLs specific for a complex containing HLA-A24 antigen efficiently proliferate and destroy cancer cells. In this way, treatment or prevention of cancer is achieved. The cancer vaccine of the present invention can be used for treating or preventing cancers with an elevated expression level of WT1 gene, including blood cancers such as leukemia, myelodysplastic syndrome, multiple myeloma and malignant lymphoma, and solid cancers such as gastric cancer, colon cancer, lung cancer, breast cancer, embryonal cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer and ovarian cancer.

In this regard, as with other embodiments, the invention provides the use of a peptide according to the invention in the manufacture of a vaccine for cancer, and a method of treatment or prevention of cancer, comprising administering an effective amount of a peptide according to the invention to a cancer patient in need of treatment that is HLA-a24 positive and WT1 positive.

The cancer vaccine comprising the peptide of the present invention as an active ingredient may contain a single CTL epitope as an active ingredient, or an epitope peptide linked to another peptide (CTL epitope or helper epitope) as an active ingredient. Recently, it has been demonstrated that a number of epitope peptides in which CTL epitope peptides (antigen peptides) are linked to each other have an activity of efficiently inducing CTLs in vivo. For example, Journal of immunology 1998, 161: 3186-3194 it is described that an epitope peptide (antigen peptide) of about 30 amino acids composed of HLA-A2, -A3, -A11, B53-restricted CTL epitopes derived from cancer antigen protein, PSA linked to each other is capable of inducing CTLs specific to the relevant CTL epitopes in vivo. Likewise, it has been demonstrated that epitope peptides in which a CTL epitope and a helper epitope are linked to each other can effectively induce CTLs. When the peptide of the present invention is administered in the form of such an epitope peptide, the peptide is introduced into antigen-presenting cells and then undergoes intracellular degradation to produce respective antigen peptides that bind to HLA antigens to form a complex. The complex is presented tightly on the surface of antigen presenting cells, and then CTLs specific for the complex efficiently proliferate and destroy cancer cells. In this way, treatment or prevention of cancer is achieved.

The cancer vaccine containing the peptide of the present invention as an effective ingredient may be administered together with a pharmaceutically acceptable carrier such as an appropriate adjuvant, or may be administered in the form of a granular dose in order to effectively establish cellular immunity. To achieve this, adjuvants such as those described in the literature (Clin. Microbiol. Rev., 7: 277-289, 1994) can be used, including in particular bacterially derived components, cytokines, plant derived components, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin and polyols, polyanions, peptides and oil emulsions (emulsion formulations). Likewise, liposomal formulations, particulate formulations in which the components are associated with particles several microns in diameter, or formulations in which the components are attached to lipids are also possible.

Administration can be by, for example, intradermal, subcutaneous, intramuscular, or intravenous injection. Although the dose of the peptide of the present invention in the preparation may vary depending on the disease to be treated, the age and body weight of the patient, etc., usually 0.0001mg to 1000mg of the peptide of the present invention, preferably 0.001mg to 1000mg, more preferably 0.1mg to 10mg, is administered every several days to every several months.

(6-2) DNA vaccine containing polynucleotide encoding the peptide of the present invention or expression vector as active ingredient

Not only the above-mentioned peptide of the present invention but also a polynucleotide encoding the peptide and an expression vector containing the polynucleotide can be used as an active ingredient in a DNA vaccine for the treatment or prevention of cancer. In a specific embodiment, the present invention provides a cancer vaccine (pharmaceutical composition useful as a cancer vaccine) comprising as an effective ingredient a polynucleotide encoding the peptide of the present invention, or an expression vector comprising the polynucleotide. In another embodiment, the present invention provides a method for treating or preventing cancer, which comprises administering an effective amount of the DNA vaccine according to the present invention to a patient positive for HLA-a24 and WT 1.

Recently, it has been demonstrated that a polynucleotide encoding an epitope peptide in which a plurality of CTL epitopes (antigen peptides) are linked to each other, or a polynucleotide encoding an epitope peptide in which a CTL epitope and a helper epitope are linked to each other, has an activity of efficiently inducing CTLs in vivo. For example, Journal of Immunology1999, 162: 3915-3925 describes DNAs encoding epitope peptides linked to 6 HLA-A24-restricted antigen peptides and 3 HLA-A11-restricted antigen peptides derived from HBV, and helper epitopes (minigenes), and having CTLs effective in inducing a response to the relevant epitope in vivo.

Thus, suitable expression vectors incorporating polynucleotides prepared by ligating one or more polynucleotides encoding a peptide of the invention to each other, or by ligating a polynucleotide of the invention to a polynucleotide encoding another peptide, are useful as active ingredients in cancer vaccines.

The following method can be used to allow the polynucleotide of the present invention to be used as an active ingredient of a cancer vaccine (DNA vaccine).

The polynucleotide of the present invention may be introduced into cells using a viral vector, or according to any of other methods (Nikkei-Science, 4 months, 1994, pp.20-45; Gekkan-Yakuji, 36(1), 23-48 (1994); Jikken-Igaku-Zokan, 12(15), 1994, and references cited therein).

Examples of the method using a viral vector include a method of integrating the DNA of the present invention into a DNA or RNA virus, such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, or Sindbis virus (Sindbis virus), and introducing into a cell. Among these methods, those using retrovirus, adenovirus, adeno-associated virus, or vaccinia virus are particularly preferable.

Other methods include a method of directly injecting an expression plasmid into muscle (DNA immunization), a liposome method, a Lipofectin method, microinjection, a calcium phosphate method, and electroporation, and the DNA immunization and the liposome method are particularly preferable.

In practice, in order to allow the polynucleotide of the present invention to be used as a medicament, there are an in vivo method in which the polynucleotide is directly introduced into the body, and an in vitro method in which a cell is isolated from the human body, DNA is introduced into the cell in vitro, and the cell is reintroduced into the body (Nikkei-Science, month 4, 1994, pp.20-45; Gekkan-Yakuji, 36(1), 23-48 (1994); Jikkenn-Igaku-Zokan, 12(15), 1994; and references cited therein). More preferably in vivo.

When an in vivo approach is employed, the polynucleotide may be administered by any appropriate route, depending on the disease and condition to be treated and other factors. For example, administration can be by intravenous, intraarterial, subcutaneous, intradermal, intramuscular, and the like. In the in vivo method, the composition can be used in various dosage forms, such as solution form, and always prepared, for example, containing the polynucleotides of the invention as active ingredients in the form of injection, which can also be according to the need, adding conventional carrier. If the polynucleotide of the present invention is contained in a liposome or a membrane-fused liposome, such as Sendai virus (HVJ) -liposome, the composition may be in the form of a liposome preparation, such as a suspension, a frozen drug concentrated by centrifugation, or the like.

Although the dose of the polynucleotide of the present invention contained in the preparation may vary depending on the disease to be treated, the age and body weight of the patient, etc., 0.0001mg to 100mg, preferably 0.001mg to 10mg, of the polynucleotide of the present invention is conventionally administered every few days to every few months.

When the polynucleotide of the present invention is administered to a cancer patient, a polypeptide corresponding to the polynucleotide is efficiently expressed in antigen presenting cells. Then, the respective cancer antigen peptides produced by intracellular degradation are bound to HLA antigen peptides to form complexes, which are tightly presented on the cell surface of the antigen presenting cells. Subsequent CTLs specific for this complex proliferate efficiently and destroy cancer cells. In this way, treatment or prevention of cancer is achieved. The cancer vaccine of the present invention, which comprises the polynucleotide of the present invention or an expression vector comprising the polynucleotide as an active ingredient, can be used for treating or preventing cancers having an increased expression level of WT1 gene, including blood cancers such as leukemia, myelodysplastic syndrome, multiple myeloma and malignant lymphoma, and solid cancers such as gastric cancer, colon cancer, lung cancer, breast cancer, embryonal cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer and ovarian cancer.

(6-3) cancer vaccine comprising the antigen-presenting cell of the present invention as an active ingredient

The present invention provides a cancer vaccine comprising the antigen-presenting cell of the present invention as an active ingredient.

Recently, there has been a report of cell therapy (DC therapy) in which lymphocytes are isolated from peripheral blood of a Cancer patient, and dendritic cells induced by the lymphocytes are pulsed in vitro with a substance such as peptide to prepare antigen-presenting cells, which are then delivered back to the patient by subcutaneous injection or other means (Cancer Immunol.Immunother., 46: 82, 1998, J.Immunol., 158: p1796, 1997, Cancer Res., 59: p1184, 1999, Cancer Res., 56: p5672, 1996, J.Immunol., 161: p5607, 1998, J.Exp.Med., 184: p465, 1996). Therefore, a cancer vaccine containing the antigen-presenting cells of the present invention as an active ingredient can be used as an active ingredient of a cancer vaccine employed in cell therapy.

The cancer vaccine containing the antigen-presenting cells of the present invention as an active ingredient preferably contains physiological saline, Phosphate Buffered Saline (PBS), a culture medium, or the like to stably maintain the antigen-presenting cells. It can be administered, for example, intravenously, subcutaneously, or intradermally. The dosages are, for example, those described in the abovementioned documents.

By reintroducing the cancer vaccine into the body of the patient, specific CTLs can be efficiently induced in HLA-A24-positive and WT 1-positive patients to thereby achieve treatment or prevention of cancer. The cancer vaccine comprising the antigen-presenting cell of the present invention as an active ingredient can be used for treating or preventing cancers having an increased expression level of WT1 gene, including blood cancers such as leukemia, myelodysplastic syndrome, multiple myeloma and malignant lymphoma, and solid cancers such as gastric cancer, colon cancer, lung cancer, breast cancer, embryonal cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer and ovarian cancer.

(6-4) cancer vaccine comprising the CTL of the present invention as an active ingredient

The present invention provides a cancer vaccine (pharmaceutical composition useful as a cancer vaccine) comprising the CTL of the present invention as an active ingredient. The CTLs of the invention are useful in the immunotherapies employed hereinafter.

For melanoma, it has been observed that certain therapeutic effects are achieved by immunotherapy using large in vitro cultures of tumor-infiltrating T cells obtained from the patient himself/herself, which are then reinfused back into the patient (J.Natl.cancer.Inst., 86: 1159, 1994). Similarly, in mouse melanoma, inhibition of metabolism has been observed by stimulating spleen cells with a cancer antigen peptide TRP-2 in vitro, thereby proliferating CTLs specific for the cancer antigen peptide, and administering the CTLs to mice transplanted with melanoma (J.Exp.Med., 185: 453, 1997). This is due to the in vitro proliferation of CTLs that specifically recognize a complex formed between an HLA antigen and a cancer antigen peptide on antigen presenting cells. Therefore, a method for treating cancer comprising stimulating peripheral blood lymphocytes of a patient in vitro using the peptide, or the polynucleotide or the expression vector as described in the present invention to proliferate tumor-specific CTLs and then delivering the CTLs back to the patient is considered to be effective. Therefore, the CTLs of the present invention can be used as an active ingredient contained in a cancer vaccine employed in the immunotherapy employed.

The cancer vaccine comprising the CTLs of the present invention as an active ingredient preferably contains physiological saline, Phosphate Buffered Saline (PBS), a culture medium, or the like to stably maintain the CTLs. It can be administered, for example, intravenously, subcutaneously, or intradermally. The dosages are, for example, those described in the above-mentioned documents.

The cytotoxic effect of CTLs on cancer cells was enhanced and cancer cells were destroyed in HLA-a 24-positive and WT 1-positive patients by reintroducing cancer vaccines into patients, thereby achieving treatment of cancer. Cancer vaccines containing the CTLs of the present invention as an active ingredient can be used for treating or preventing cancers with an increased level of expression of WT1 gene, including blood cancers such as leukemia, myelodysplastic syndrome, multiple myeloma, and malignant lymphoma, and solid cancers such as gastric cancer, colon cancer, lung cancer, breast cancer, embryonal cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, and ovarian cancer.

(VII) comprises a sequence based on SEQ ID NO: 7 amino acid sequence of peptides for cancer vaccines

In the present invention, it has been demonstrated that a peptide having the amino acid sequence Arg Val Pro Gly Val Ala Pro ThrLeu (SEQ ID NO: 7) has an activity of inducing CTLs in vivo. In WO00/18795, the polypeptide encoded by SEQ ID NO: 7 amino acid sequence is described as a peptide having a sequence expected to bind to HLA-a24 antigen. However, in the present invention, it was first found that the peptide has an activity of inducing CTLs in vivo and can be used as a cancer vaccine.

Accordingly, the present invention provides a pharmaceutical composition or a cancer vaccine comprising any one selected from the group consisting of:

a) comprises the amino acid sequence shown in SEQ ID NO: 7 amino acid sequence of a peptide having a sequence,

b) a polynucleotide encoding the peptide of a) above,

c) an expression vector comprising the polynucleotide of b) above,

d) a cell comprising the expression vector of c) above,

e) an antigen-presenting cell which presents on its surface a complex formed between a cancer antigen peptide derived from the peptide represented by a) above and HLA-A24 antigen, and

f) CTLs recognizing a complex formed between a cancer antigen peptide derived from the peptide represented by a) above and HLA-A24 antigen.

Furthermore, the present invention also provides the use of any of the above-mentioned peptides, polynucleotides, expression vectors, transformants, antigen-presenting cells and CTLs for the preparation of a cancer vaccine, and a method for the treatment or prevention of cancer, which comprises administering a therapeutically or prophylactically effective amount of any of those to a cancer patient who is HLA-a24 positive and WT1 positive in need of treatment.

The methods for preparing the substances described in a) to f) above, and their use as cancer vaccines are the same as described for the peptide, polynucleotide, expression vector, antigen presenting cell and CTL of the present invention.

Examples

The present invention is further illustrated by the following examples, but is not limited in any way by these examples.

A method for preparing a transgenic mouse expressing HLA-A24 is described below with reference to the protocol described in detail in WO 02/47474 (International publication date: 6/20/2002, PCT/JP01/10885 (International application date: 12/2001, (priority date: 12/13/2000))).

Reference 1

Cloning of HLA-A2402 genomic DNA fragment

(1) Cloning of HLA-A2402 genomic DNA fragment

For the purpose of cloning human HLA-A2402 genomic DNA by PCR, a human tumor Cell line, RERF-LC-AI cells (Riken Cell Bank RCB0444) was cultured, and human genomic DNA was purified according to the attached protocol using genomic Prep Cell and tissue DNA isolation kit (Amersham). HLA-A2402 genomic DNA required for construction of the chimeric HLA gene was then searched in GeneBank database showing that the sequence of accession No. Z72422 was related but a 270bp promoter region was not registered. The construction of transgenic mice of interest requires a promoter, exons 1 to 3 and introns 1 to 3. To clone HLA-A2402 genomic DNA containing a promoter, PCR was performed using an upstream primer, HLA 26-1F: 5'-CCC AAG CTT ACT CTC TGG CAC CAA ACT CCA TGG GAT-3' (36 nucleotides, SEQ ID NO: 36), which is designed with reference to the nucleotide sequence of HLA-A2601 promoter (accession No. AB005048) frequently found in Japan; downstream primer, a24-Bgl II 30: 5'-CGG GAG ATC TAC AGG CGA TCA GGT AGG CGC-3' (30 bases, SEQ ID NO: 37), which primer contains a modification in the intron 3 nucleotide sequence, specifically, the nucleotide at position 1282 of the 5 ' end of accession No. Z72422 from G to A.

The nucleotide needs to be modified for the following reasons. The reference scheme is aimed at obtaining exons 1-3 and H-2K expressed from HLA-A2402^bThe chimeric HLA transgenic mouse consisting of exons 4 to 8 of (A) can be obtained by ligating the upstream region of the BamHI cleavage site in intron 3 of HLA-A2402 genomic DNA and H-2K^bThe downstream region of intron 3 of genomic DNA was ligated to construct a chimeric HLA, and for this end, an artificial BglII cleavage site had to be constructed in intron 3 of HLA-A2402.

The HLA-a2402 genomic DNA fragment was then PCR cloned using native Pfu DNA polymerase (Stratagene) with high 3 '→ 5' exonuclease activity and the above primer pair according to the attached protocol. The PCR reaction included a 95 ℃ heat treatment for 45 seconds, 35 reaction cycles, each cycle: 95 ℃ for 45 seconds, 66 ℃ for 1 minute, 72 ℃ for 4 minutes, and 72 ℃ for 10 minutes, followed by cooling to 4 ℃. The amplified gene fragment was ligated to a phagemid vector, HindIII and BamHI cleavage sites of pBluescript to obtain a recombinant plasmid. The recombinant plasmid was introduced into Escherichia coli JM109(Toyobo) at 42 ℃ by the heat shock method, and white colonies of those Escherichia coli into which the recombinant plasmid had been introduced were selected on LB agar medium (1% bacto tryptone, 0.5% yeast extract, 1% sodium chloride, 2% agar) containing ampicillin (50. mu.g/ml) coated with X-Gal and IPTG to obtain transformants.

(2) Determination of nucleotide sequence of HLA-A2402 promoter region

The 4 transformants obtained IN the above-mentioned method were cultured overnight IN LB medium (3ml) containing ampicillin, and then the plasmid clone contained IN each transformant was purified by alkaline lysis (Current promoters IN MOLECULAR BIOLOGY, ed. F.M.Ausubel et al, John Wiley&Sons, Inc.). Then through ABI PRISM^TM377 DNA sequencing System (PE biosystem) determines its nucleotide sequence. According to the attached scheme, ABI PRISM is used^TMStaining termination cycle sequencing preliminary reaction kit (ABI PRISM)^TMDyeTerminator Cycle Sequencing Ready Reaction kit (PE biosystems) Sequencing samples were sequenced to determine the sequence of each clone. When comparing the promoters of the respective clones, the results show that they are identical. Thus, the nucleotide sequence of HLA-A2402 promoter region has been determined and has not been registered in the GenBank database. Comparing the nucleotide sequence of accession No. z72422 with the nucleotide sequences of the respective clones, it was revealed that there were 1 normal clones without PCR mutation.

Reference 2

Clone H-2K^bGenomic DNA fragments

(1)H-2K^bCloning of genomic DNA fragments

The mouse tumor cell line EL4(ATCC T1B-39) was cultured, and mouse genomic DNA was purified and used for PCR cloning. Use of TaKaRa LA Taq suitable for amplification of long-chain DNA^TM(Takara Shuzo), purification of DNA was carried out according to the attached protocol. Then search GenBank database for H-2K required for constructing chimeric HLA gene^bGene, the results showed that the gene was divided into two fragments with accession numbers nos. v00746 and v 00747. H-2K^bThe upstream 1594bp region upstream of intron 3 (b) was registered as v00746, H-2K^bThe downstream 1837bp region of intron 7 (b) was registered as v 00747. Because intron 3 has no BamHI cleavage site, H-2K was segmented and registered as v00746 and v00747 in the database^bGenes, considered to be incomplete in length.

In H-2K^bAmong the genes are homologous pseudogenes or highly homologous genes (cell., 25: 683, 1981). TaKaRa LA Taq was used according to the attached protocol^TM(Takara Shuzo) and PCR was performed using the forward primer H-2KB F3: 5'-CGC AGG CTC TCA CAC TAT TCA GGTGAT CTC-3' (30 bases, SEQ ID NO: 38) which has low homology to the complementary gene and is encoded by exon 3 of v 00746; downstream primer H-2KB 3R: 5'-CGGAAT TCC GAG TCT CTG ATC TTT AGC CCT GGG GGC TC-3' (38 bases, SEQ ID NO: 39) corresponding to v00747 with an EcoRI cleavage site added to the end, and the above-mentioned purified mouse genomic DNA was used as a template. PCR consisted of 25 reaction cycles, 98 ℃ for 10 seconds, 66 ℃ for 4 minutes, and 68 ℃ for 10 minutes, followed by cooling to 4 ℃.

The amplified gene fragment was ligated to the restriction sites KpnI and EcoRI of the phagemid vector pBluescrip t to obtain a recombinant plasmid. The recombinant plasmid was introduced into Escherichia coli JM109(Toyobo) at 42 ℃ by the heat shock method, and white colonies of those Escherichia coli into which the recombinant plasmid had been introduced were selected on LB agar medium containing ampicillin coated with X-Gal and IPTG, to obtain transformants. 3 transformants were cultured overnight in LB medium (3ml) containing ampicillin. Plasmid clones contained in each transformant were purified, and nucleotide sequence analysis was performed in a similar manner as described above. A comparison of the nucleotide sequences of 3 clones and v00747 showed that there was one PCR mutation in each of the 2 clones and 3 PCR mutations in the other clone. There were 5 commonly found nucleotides in these 3 clones, which were different from those of v 00747. These nucleotides are found in the region corresponding to intron 6 and the 3' non-coding region. In addition, the unregistered intron 3 region contained nucleotides obtained by PCR mutation different from those in 3 clones. It is thus to some extent impossible to determine the nucleotide sequence for the unregistered region, which can be obtained after recloning the unregistered intron 3 region using a polymerase with high 3 '→ 5' exonuclease activity.

(2)H-2K^bDetermination of intron 3 nucleotide sequence

To determine the nucleotide sequence of the unregistered region, a region containing the unregistered intron 3 region was cloned by PCR using purified mouse genomic DNA as a template and a natural Pfu DNA polymerase (Stratagene) according to the attached protocol. The PCR reaction was performed using the forward primer H-2kbF 5: 5'-AGG ACT TGG ACT CTG AGA GGC AGG GTC TT-3' (29 bases, SEQ ID NO: 40), having accession number v 00746; downstream primer H-2kb 5R: 5'-CAT AGT CCC CTC CTT TTC CAC CTG TGA GAA-3' (30 bases, SEQ ID NO: 41), which was registered as v 00747. PCR consisted of a 95 ℃ heat treatment for 45 seconds, 25 reaction cycles, each cycle being: 95 ℃ for 45 seconds, 68 ℃ for 1 minute, 72 ℃ for 4 minutes, and reaction at 72 ℃ for 10 minutes, followed by cooling to 4 ℃. The amplified gene fragment was ligated to the BamHI and BglII cleavage sites of the phagemid vector pBluescript to obtain a recombinant plasmid. The recombinant plasmid was introduced into Escherichia coli JM109(Toyobo) at 42 ℃ by the heat shock method, and white colonies of Escherichia coli into which the recombinant plasmid had been introduced were selected on LB agar medium (1% bacto tryptone, 0.5% yeast extract, 1% sodium chloride, 2% agar) containing ampicillin (50. mu.g/ml) coated with X-Gal and IPTG to obtain transformants. 5 transformants were cultured overnight in LB medium (3ml) containing ampicillin, and the plasmid clone contained in each transformant was purified and analyzed for nucleotide sequence in a similar manner as described above. Comparison of the intron 3 regions of the clones analyzed, respectively, revealed that the sequences were identical. Thus, the nucleotide sequence of the intron 3 region was determined. In addition, the region from the BamHI site in the unregistered region to v00747 was shown to be 463 bp.

(3)H-2K^bConstruction of genomic DNA

By determining the nucleotide sequence not registered in (2), H-2K necessary for constructing the desired chimeric HLA gene was identified^bThe entire nucleotide sequence of genomic DNA. It became clear that by combining the two clones obtained as described above, i.e.,in the 5 '-and 3' -regions, respectively, H-2K without PCR mutation^b#20 and H-2K without PCR mutation^b#26 the objective H-2K can be constructed^bGenomic DNA. Therefore, the PCR mutation-free H-2K was constructed by cleaving these clones with restriction enzymes and combining the respective PCR mutation-free regions^bGenomic DNA. A schematic of the construction is shown in figure 1.

Both clones were cleaved at the BglII and EcoRI cleavage sites and ligated to obtain a recombinant plasmid. The recombinant plasmid was introduced into Escherichia coli JM109(Toyobo) at 42 ℃ by the heat shock method, and white colonies of Escherichia coli into which the recombinant plasmid had been introduced were selected on LB agar medium containing ampicillin coated with X-Gal and IPTG to obtain transformants. 3 transformants were cultured overnight in LB medium (3ml) containing ampicillin. The plasmid clone contained in each transformant was purified by an alkaline lysis method, and sequence analysis was performed by the similar method as described above. As a result, sequence analysis showed that all transformants contained H-2K encoding no PCR mutation^bPlasmids of genomic DNA.

H-2K obtained here^bThe nucleotide sequence of the genomic DNA corresponds to a sequence selected from the group consisting of SEQ id nos: 33, the 1551 th nucleotide downstream nucleotide sequence, which will be described below.

Reference 3

Chimeric genomic DNA (HLA-A2402/K)^bDNA) construction

The plasmid HLA-A2402#1 containing HLA-A2402 genomic DNA obtained in the above reference scheme 1 was cleaved at the BglII cleavage site, and the plasmid HLA-A2402#1 containing H-2K obtained in the above reference scheme 2 was cleaved at the BamHI cleavage site^bPlasmid H-2K of genomic DNA^b#20/26, and ligating the resulting fragments to obtain a recombinant plasmid. A schematic of the construction is shown in figure 2. The recombinant plasmid was introduced into Escherichia coli JM109(Toyobo) at 42 ℃ by the heat shock method, and white colonies of Escherichia coli into which the recombinant plasmid had been introduced were selected on LB agar medium containing ampicillin coated with X-Gal and IPTG to obtain transformants. In the presence of ampicillin10 transformants were cultured overnight in LB medium (3ml) for penicillin. Plasmid clones contained in each transformant were purified and subjected to sequence analysis in a similar manner as described above. As a result, sequence analysis revealed that 3 transformants contained HLA-A2402/K carrying the objective chimeric gene^bPlasmid of DNA, which may be simply referred to as "A2402/K^bDNA ". Constructed HLA-A2402/K^bThe genome sequence of (a) is as shown in SEQ ID NO: shown at 33.

Reference 4

Splicing analysis of chimeric genomic DNA

Using Electro Gene Transfer GTE-10(Shimadzu), the constructed chimeric HLA Gene (HLA-A2402/K) was used according to the attached protocol^bGene) transfected mouse tumor cell line EL 4. After 2 days, the total RNA of transfected EL4 cells and untransfected EL4 cells was purified according to the attached protocol using ISOGEN (Nippon Gene). Using the SuperScript Choice System (GIBCO BRL), oligo (dT) was used according to the attached protocol_12-18And a part of the RNA is used as a template for reverse transcription to synthesize cDNA. In addition, the chimeric gene was specifically amplified by PCR using native Pfu DNA polymerase (Stratagene) and a portion of the cDNA as a template.

PCR reactions were performed using the forward primer chimera-F2: 5'-CGA ACC CTC GTC CTGCTA CTC TC-3' (23 bases, SEQ ID NO: 42) encoded in exon 1 of HLA-A2402 gene and associated with H-2K^bThe genes have lower homology, and the downstream primer chimera-R2: 5'-AGC ATA GTC CCC TCC TTT TCC AC-3' (23 bases, SEQ ID NO: 43) at H-2K^bThe gene is encoded in exon 8 and has low homology with HLA-A2402 gene, and the reaction conditions are as follows: heat treatment at 95 ℃ for 45 seconds, 40 reaction cycles, each cycle being: 95 ℃ for 45 seconds, 53 ℃ for 1 minute, and 72 ℃ for 2 minutes, and at 72 ℃ for 10 minutes, followed by cooling to 4 ℃.

As a result, a gene fragment of about 1.1kb was specifically amplified only in transfected EL4 cells. Based on this result, the transfer can be estimatedThe chimeric genomic DNA is transcribed in mouse cells, i.e., the HLA promoter functions, and expression of mRNA spliced at the predicted position occurs. Sequencing of the fragment amplified by the above PCR, as expected, identified the coding HLA-A2402/K^bThe nucleotide sequence of cDNA of (1). Encoding said HLA-A2402/K^bThe base sequence of the cDNA of (1) is shown in SEQ ID NO: 34, as shown in SEQ ID NO: the amino acid sequence is shown in 35. Furthermore, FIGS. 3 to 5 show HLA-A2402/K arranged in parallel^bThe genomic sequence (SEQ ID NO: 33) and the cDNA sequence (SEQ ID NO: 34).

Reference 5

Preparation of DNA solution for microinjection

The plasmid (11. mu.g) encoding the constructed chimeric HLA gene was digested with restriction enzymes HindIII and EcoRI, and similarly digested with restriction enzyme DraI which cleaves only the vector. After gel electrophoresis (1% SeaKem GTG, Nippon Gene), gel fragments containing the chimeric DNA were recovered. A DNA solution for microinjection was prepared by purifying the transgene according to the attached protocol using a Prep-A-gene purification kit (BioRad) and dissolving in 1/10TE buffer (10mM Tris (pH 8), 0.1mM EDTA (pH 8)).

Reference 6

Introduction into mouse fertilized eggs and identification of transgenic mice

The chimeric gene construct was injected using fertilized eggs from the C57BL/6 mouse line.

Since C57BL/6 mice express H-2b as a class I molecule rather than H-2K with a similar binding motif for HLA-A2402^bTherefore, fertilized eggs of the C57BL/6 mouse line were used. Thus, the C57BL/6 line transgenic mice advantageously avoid cross-reactivity when administered HLA-a24 restricted antigenic peptides, since class I endogenous mice do not present the peptides on the cell surface.

In the first injection, the chimeric construct was injected into 81 fertilized eggs, which were transferred to 4 recipient mice, resulting in no childbirth. In the second injection, the chimeric construct was injected into 50 fertilized eggs, which were transferred to 2 recipient mice, resulting in the delivery of 4 offspring, but all of which died before weaning. In the third injection, the chimeric construct was injected into 101 fertilized eggs, which were transferred to 4 recipient mice, resulting in the delivery of 11 offspring, but all of which died before weaning.

In the fourth injection, the chimeric construct was injected into 168 fertilized eggs, which were transferred to 6 recipient mice, resulting in a delivery that yielded 22 offspring, 19 of which were discontinued from lactation. 4 of these, 01-4, 04-2, 05-1 and 05-6, were identified as transgenic mice; however, since the malformed 01-4 mice were unable to mate, the 05-6 mice died soon after weaning. In the fifth injection, the chimeric construct was injected into 221 fertilized eggs, which were transferred to 8 recipient mice, resulting in the delivery of 14 offspring, 6 of which were weaned from lactation. 3 of these, 04-1, 04-5 and 04-6, were identified as transgenic mice. In the sixth injection, the chimeric construct was injected into 225 fertilized eggs, which were transferred to 8 recipient mice, resulting in the delivery of 13 offspring, 9 of which were weaned from lactation. 3 of these, 10-5, 14-1 and 15-2, were identified as transgenic mice.

By using TaKaRa LA Taq^TM(Takara Shuzo), as described in the attached protocol, PCR was performed using the same primers (HLA26-1F, SEQ ID NO: 36; and A24-BglII30, SEQ ID NO: 37) and tail DNA preparations as those used for cloning HLA-A2402 gene as templates, 1% agarose gel electrophoresis was applied, and mice were selected based on the presence of 1.5kbp DNA band.

Reference 7

Expression of transgenic products in transgenic mice

Edited by CURRENT PROTOCOLS IN IMMUNOLOGY, J.E.Coliganl et al, John Wiley&Method described in Sons, IncSpleen cells were recovered from spleens isolated from mice of 8 transgenic mouse lines 04-2, 05-1, 04-5, 04-6, 10-5, 14-1 and 15-2 constructed in reference scheme 6. Analysis of the protein derived from the transgene, HLA-A2402/K, by flow cytometry^bExpression on the cell surface of splenocytes from transgenic mice. Splenocytes prepared from the C57BL/6 line were used as controls. Specifically, 5 × 10⁶Individual splenocytes were stained with FITC-labeled anti-HLA antibody B9.12.1 (Immunotech). FITC-labeled anti-H-2K of I-class endogenous mouse^bMonoclonal antibody AF6-88.5 (Pharmingen).

As a result, 5 lines, namely, 04-1, 04-5, 10-5, 14-1 and 15-2 showed specific expression for HLA class I. Among them, only line 04-1 showed the reproductive ability. On the other hand, the other 3 germline, i.e., 04-6, 04-2 and 05-1, showed no specific expression for class I HLA. Thus, 8 transgenic mice were constructed, however, only 04-1 line among them showed class I expression pattern and obtained homozygosity.

Reference 8

Establishment of HLA-A2402-expressing transformed cells

In order to evaluate the CTL-inducing ability of the transgenic mice prepared as described above, stably expressed HLA-2402/K was established^bThe transformed cell Jurkat-A2402/K^b。

(1) Construction of expression vectors

Spleens were isolated from Tg mice and splenocytes were prepared. Total RNA was prepared according to the attached protocol using isogen (nippon gene). Using the SuperScript selection System (GIBCO BRL), according to the attached protocol, oligo (dT)_12-18And a part of the RNA is used as a template, reverse transcription is carried out, and cDNA is synthesized. Then, by using a LA-PCR kit (Takara Shuzo), a part of the cDNA as a template, and an upstream primer chi. pf1: 5'-CCC AAG CTT CGC CGA GGA TGG CCG TCA TGG CGC CCCGAA-3' (SEQ ID NO: 44); and downstream primer chi. pr1: 5' -CCG GAA TTC TGTCTT CAC GCT AGA GAA TGA GGG TCA TGAAC-3' (SEQ ID NO: 45) was subjected to PCR. PCR consisted of a 95 ℃ heat treatment for 45 seconds, 25 reaction cycles, each cycle being: 95 ℃ for 45 seconds, 60 ℃ for 1 minute, and 68 ℃ for 2 minutes, and 72 ℃ for 10 minutes, followed by cooling to 4 ℃. The PCR-amplified gene was introduced into an expression vector pcDNA3.1(+) (Invitrogen) to construct a gene encoding HLA-A2402/K^bThe expression vector of (1).

(2) Introduced into Jurkat cells

The vector (10. mu.g) was linearized by digestion with the PvuI restriction enzyme. Using a Gene transfer device (GIBCO BRL), the constructed chimeric HLA gene was transfected by 5X 10 according to the attached protocol⁶Jurkat cells (ATCC T1B-152). Cells were seeded at a density of 0.5 cells/well in 96-well plates and cultured in medium containing Genetic (0.6 mg/ml). As a result, cell proliferation was observed in 6 wells (6 clones, A-2, A-4, A-6, A-9, A-10 and A-11). Wherein A-10 showed the highest expression of the transgene and the clone Jurkat-A2402/K was established^bA cell.

Reference 9

Test of CTL-inducing ability in transgenic mice

The human tumor antigen HER-2/neu is known to be overexpressed in breast, ovarian and lung cancers, and in vitro experiments show that peptides derived therefrom have activity in inducing specific CTLs in the peripheral blood of HLA-A24 positive healthy subjects (int.J. cancer., 87: 553, 2000).

Using HLA-restricted peptide HER-2/neu derived from said human tumor antigen_780-788(SEQ ID NO: 46) and MHC class II I-A derived from tetanus toxin^bTransgenic mice were immunized with a restriction helper peptide (Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys ValSer Ala Ser His Leu Glu; SEQ ID NO: 32) and examined whether specific CTLs were induced as in the case of immunization with human. Specifically, HER-2/neu_780-788And the helper peptide was adjusted to 40mg/ml and 20mg/ml in DMSO and diluted to 2mg/ml and 1mg/ml in physiological saline, respectively. Use glassGlass syringes were mixed with equal amounts of Freund's incomplete adjuvant (Wako Pure Chemical Industries, Ltd.) to prepare water-in-oil emulsions. The resulting preparation (200. mu.l) was injected subcutaneously into transgenic mice (line 04-1) at the tail root for immunization. At 7 days after the start of the experiment, spleens were isolated and ground on a frosted portion of a glass slide, and splenocytes were recovered and prepared. Will use ACK buffer (0.15M NH)₄Cl，10mM KHCO₃0.1mM EDTA, pH 7.2-7.4) A portion of the hemolyzed splenocytes was exposed to X-ray radiation (2,000 rads), pulsed with the above peptide (100. mu.g/ml) for 1 hour at 0.7X 10⁶Density per well was planted in 24 well plates. Splenocytes (7X 10) were added together without irradiation and without peptide pulse treatment⁶Perwell) and restimulated at 37 deg.C (final concentration of peptide, 1. mu.g/ml). In vitro stimulation was performed for 6 days in 10ml of a culture medium RPMI1640 containing 10% FCS, 10mM HEPES, 20mM L-glutamine, 1mM sodium pyruvate, 1mM MEM non-essential amino acids, 1% MEM vitamins and 55. mu.M 2-mercaptoethanol (CTM culture medium).

On the other hand, Jurkat-A2402/K prepared in reference scheme 8^bFor cells⁵¹Cr(3.7MBq/10⁶Cells) and pulsed with the above peptide at a concentration of 100. mu.g/ml for 1 hour. Labeling was carried out for 2 hours or more, and 1 hour after the start of labeling, peptide was added to a final concentration of 100. mu.l/ml. Cells that were not treated with peptide pulses were prepared as control target cells.

By passing⁵¹The Cr release assay measures CTL-inducing activity (J.Immunol., 159: 4753, 1997) in which the Jurkat-A2402/K is administered to target cells^bThe cells were added with a pre-prepared preparation of splenocytes from transgenic mice. The results are shown in fig. 6. As a result, the passage through HER-2/neu was observed_780-788The stimulation of (2) induces specific CTLs.

In addition, MAGE-3 was used_195-203(SEQ ID NO：47)、CEA_652-660(SEQ ID NO: 48) and CEA_268-277(SEQ ID NO: 49), CTL-inducing ability was examined in the same manner, and it is known that these peptides are also like HER-2/neu_780-788The same HLA-A24-restricted cancer antigen peptide. The results are shown in figures 7 to 9. As a result, it was observed that specific CTLs were induced by the stimulation with these known HLA-A24-restricted cancer antigen peptides.

These results show that the HLA-A24 transgenic mouse of the present invention is an animal model for human use which can be used for in vitro evaluation of HLA-A24-restricted cancer antigen protein or cancer antigen peptide.

Example 1

Natural peptide derived from human WT1 and CTL-inducing activity of altered peptide

The amino acid sequence of human WT1 was searched using BIMAS software (http:// BIMAS. dcrt. nih. gov/molbio/HLA _ bind /) for sequences that were predicted to bind HLA-A24 antigen. This search identified the following peptides:

peptide A: arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 8),

peptide B: arg Val Pro Gly Val Ala Pro Thr Leu (SEQ ID NO: 7),

peptide C: arg Trp Pro Ser Cys Gln Lys Lys Phe (SEQ ID NO: 9),

peptide D: gln Tyr Arg Ile His Thr His Gly Val Phe (SEQ ID NO: 10) and

peptide E: ala Tyr Pro Gly Cys Asn Lys Arg Tyr Phe (SEQ ID NO: 11).

Peptides A, B, C, D and E correspond to the sequences at positions 126 to 134, 302 to 310, 417 to 425, 285 to 294, and 326 to 335, respectively, of the amino acid sequence of human WT 1. These peptides were synthesized using the Fmoc method.

The following variant peptides were also synthesized using the Fmoc method, among others in native form: in peptides a to C, the amino acid variation at position 2 is tyrosine:

peptide F: arg Tyr Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 2),

peptide G: arg Tyr Pro Gly Val Ala Pro Thr Leu (SEQ ID NO: 3) and

peptide H: arg Tyr Pro Ser Cys Gln Lys Lys Phe (SEQ ID NO: 4).

Using HLA-2402/K constructed in the above reference protocol^bTransgenic mice were evaluated for immunogenicity of each antigenic peptide. To evaluate the immunogenicity of each peptide, 3 transgenic mice were immunized with one peptide.

Combination of tetanus toxin-derived mouse MHC class II I-A with each synthetic peptide^bThe transgenic mice were immunized with the restriction helper peptide (Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val ProLys Val Ser Ala Ser His Leu Glu; SEQ ID NO: 32). Specifically, each of the antigenic peptide and the helper peptide was adjusted to 40mg/ml and 20mg/ml in DMSO and diluted to 2mg/ml and 1mg/ml with physiological saline, respectively. They were mixed with an equal amount of Freund's incomplete adjuvant (IFA) using a glass syringe to prepare water-in-oil emulsions. The resulting emulsion (200. mu.l) was injected subcutaneously into HLA-A2402/K at the tail root^bImmunization was performed in transgenic mice. At 7 days after the start of the experiment, spleens were isolated and ground on a frosted portion of a glass slide, and splenocytes were recovered and prepared. Will use ACK buffer (0.15M NH)₄Cl，10mM KHCO₃0.1mM EDTA, pH 7.2-7.4) A fraction of hemolyzed splenocytes exposed to X-ray radiation (2,000 rads) and then pulsed with the above peptide (100. mu.g/ml) for 1 hour at 7X 10⁶Density per well was planted in 24 well plates. At the same time, non-irradiated, non-peptide-pulsed splenocytes (7X 10 cells) were added together⁵Per well) was stimulated in vitro at 37 ℃ for 6 days. In vitro stimulation was performed in RPMI1640 medium supplemented with 10% FCS, 10mM HEPES, 20mM L-glutamine, 1mM sodium pyruvate, 1mM MEM non-essential amino acids, 1% MEM vitamins and 55. mu.M 2-mercaptoethanol.

Next, the cytotoxic activity was examined according to a conventional method. Jurkat-A2402/K to be pulsed with peptide^bCells (see scheme 8) and Jurkat-A2402/K^bThe cells serve as target cells (T). By using⁵¹Cr(3.7MBq/10⁶Cells) and pulsed with peptide at a concentration of 100. mu.g/ml for 1 hour (labeling was performed for more than 2 hours, 1 hour after the start of labeling, peptide was added). Splenocytes stimulated and cultured in vitro were used as effector cells (E). Combining and reacting them at an E/T ratio of 80 by⁵¹The Cr release assay measures cytotoxic activity (j. immunol., 159: 4753, 1997). The results are shown in FIGS. 10 to 17. The Y-axis indicates cytotoxic activity and the numbers 1, 2 and 3 on the X-axis indicate the number of 3 mice.

These figures show that of the 5 WT1 native peptides tested above, only peptide B was immunogenic. The natural type: modified form of amino acid at position 2 of peptide B to tyrosine: peptide G, shown to be more immunogenic than peptide B. Likewise, although the natural type: peptides a and C are not immunogenic, but are naturally: altered versions of amino acids at position 2 of peptides a and C to tyrosine: peptides F and H, shown to be highly immunogenic.

These results demonstrate that native peptide B and altered peptides F, G and H exhibit the function of antigenic peptides that induce CTLs activity in vivo.

Example 2

Induction Activity of CTL derived from human WT1 altered peptide (II)

The following natural peptides (peptides K and L) having a sequence predicted to bind to HLA-a24 antigen, which were searched and identified using the BIMAS software, and variant peptides (peptides I and J) in which the 2 nd amino acid in the natural type was mutated to tyrosine, were synthesized using the Fmoc method in a similar manner to example 1:

peptide K: ala Leu Leu Pro Ala Val Pro Ser Leu (SEQ ID NO: 51),

peptide L: asn Gln Met Asn Leu Gly Ala Thr Leu (SEQ ID NO: 52),

peptide I: ala Tyr Leu Pro Ala Val Pro Ser Leu (SEQ ID NO: 5), and

peptide J: asn Tyr Met Asn Leu Gly Ala Thr Leu (SEQ ID NO: 6).

Peptides K and L correspond to the sequences at positions 10 to 18 and 239 to 247, respectively, of the amino acid sequence of human WT 1. Peptides I and J are altered peptides in which the amino acid at position 2 in peptides K and L, respectively, is changed to tyrosine. The immunogenicity of each of these native and altered peptides was evaluated in a similar manner to example 1. The results are shown in FIGS. 18, 19, 21 and 22. The Y-axis indicates cytotoxic activity and the numbers 1, 2 and 3 in the X-axis indicate the number of 3 mice.

These figures show that, although native peptides K and L are not immunogenic, modified peptides I and J are both highly immunogenic.

The results demonstrate that WT1 is modified: peptides I and J were shown to have the function of antigenic peptides to induce cytotoxic T cells in vivo.

Example 3

Cytotoxic Activity of altered peptide derived from human WT1

The effector cells induced by the altered peptide were tested for cross-reactivity to the native peptide. Effector cells induced by immunization of mice with altered peptide H (E) and treatment of Jurkat-A2402/K with native peptide C^bTarget cells (T) obtained from the cells, bound and reacted at an E/T ratio of 80, by⁵¹The Cr release assay measures cytotoxic activity. The results are shown in fig. 20. The figure shows that effector cells induced by WT1 altered peptides show cytotoxic activity against cells pulsed with the altered and native peptides.

Example 4

CTL induction from human peripheral blood mononuclear cells by altered peptide derived from human WT1

Isolation of peripheral blood mononuclear cells from HLA-A2402-positive healthy donors at 4X 10⁶The density of cells/well is placed in the wells of a 24-well plate. Add 10. mu.M concentration of SEQ ID NO: 7 or the native peptide of SEQ ID NO: 3, the mixture was cultured in a medium containing 45% RPMI1640, 45% AIV, 10% inactivated human AB serum, 1 × non-essential amino acids, 25ng/ml 2-mercaptoethanol, 50mg/ml streptomycin and 50U/ml penicillin for one week. After culturing, the cells were adjusted to 2X 10⁶Cells/well, used as responder cells hereinafter. On the other hand, peripheral blood mononuclear cells isolated from the same healthy donor were cultured with 10. mu.M of any of these peptides for 4 hours to complete the pulse with the peptide, followed by irradiation at 30 Gy. These cells were adjusted to 4X 10⁶Cells/well, used as stimulating cells hereinafter.

The responder cells and the stimulator cells thus prepared were mixed together, and IL-2 was added to the mixture at a concentration of 30U/ml and cultured. Similar stimulation was performed with the stimulated cells on the responding cells 3 times over a one week interval. By passing⁵¹Determining the cytotoxic activity of those cells thus obtained by a Cr release assay⁵¹Cr-labeled, with SEQ ID NO: 7C 1R-a 2402 cells positive for HLA-a24 pulsed with the native peptide of SEQ ID NO: 7 or the above-described SEQ ID NO: 3 (effector cells) (E), the cytotoxic activity was measured in response to an E/T ratio of 10, 20 or 40. The results are shown in fig. 23. This figure shows that the altered peptide can induce CTLs recognizing the native peptide and shows better activity of inducing CTLs than the native peptide. Furthermore, lung cancer cell lines positive for WT1 and positive for HLA-A24, RERF-LC-AI cells; lung cancer cell lines positive for WT1 and negative for HLA-A2402, 11-18 cells; or a lung cancer cell line negative for WT1 and positive for HLA-A24, 11-18 cells were used as target cells in a similar manner by⁵¹The Cr release assay measures the cytotoxic activity of the effector cells described above. The results are shown in fig. 24. The figure shows that the effector cells stimulated with the altered and native peptides only specifically damaged RERF-LC-AI cells positive for both WT1 and HLA-a2402, indicating that CTLs specific for WT1 and restricted by HLA-a2402 are induced by stimulation with this peptide. This tooIt was shown that the altered peptides showed better activity in inducing CTLs than the native peptides.

Example 5

CTL-inducing activity of peptide having cysteine residue substituted

Peptide H (Arg Tyr Pro Ser Cys Gln Lys Lys Phe; SEQ ID NO: 4) contains a cysteine residue at position 5. Cysteine residues may be oxidized in solution to form disulfide bonds. To avoid this, a substitution pattern in which the cysteine residue at position 5 is substituted with a serine residue, an alanine residue or α -aminobutyric acid is synthesized: peptides M, N, and O, the immunogenicity of each peptide was evaluated in vivo:

peptide M: Arg-Tyr-Pro-Ser-Ser-Gln-Lys-Lys-Phe (SEQ ID NO: 66),

peptide N: Arg-Tyr-Pro-Ser-Ala-Gln-Lys-Lys-Phe (SEQ ID NO: 67) and

peptide O: Arg-Tyr-Pro-Ser-Abu-Gln-Lys-Lys-Phe (SEQ ID NO: 68).

These substituted forms were synthesized using the Fmoc method: peptides M, N, and O, and their immunogenicity was evaluated in a similar manner to example 1. In the test of cytotoxic activity, splenocytes stimulated and cultured in vitro were used as effector cells (E) mixed in various ratios with target cells by⁵¹The Cr release assay measures cytotoxic activity of effector cells (J.Immunol., 1997; 159: 4753). The results are shown in FIGS. 25 to 28. In the figure, the vertical axis represents cytotoxic activity, and the horizontal axis represents E/T ratio.

This figure shows that peptide M, N in which the cysteine residue at position 5 in peptide H was substituted with a serine residue, an alanine residue or α -aminobutyric acid, and O have immunogenicity equivalent to that of unsubstituted peptide, peptide H.

Example 6

Cytotoxic Activity of peptides with cysteine residue substituted

The effector cells induced by the substituted peptides were tested for cross-reactivity to the unsubstituted peptides. Jurkat-A2402/K to be pulsed with peptide M or N, with peptide H, or without any peptide^bThe target cells (T) are reacted with effector cells (E) induced by immunization of mice with the peptides M or N⁵¹The Cr release assay measures cytotoxic activity of effector cells. The results are shown in fig. 29 and 30.

These figures show that effector cells induced by the substituted peptides show cytotoxic activity against all cells pulsed with the substituted peptides (peptides M and N; immune peptides in the figures) as well as the unsubstituted peptide (peptide H).

Industrial applicability

According to the present invention, WT 1-derived HLA-A24-restricted peptides having in vitro CTLs inducing activity, polynucleotides encoding the peptides, or cancer vaccines comprising the peptides or polynucleotides. The cancer vaccines of the present invention are useful in treating a number of cancer patients.

Sequence listing

<110>Haruo Sugiyama

Chugai Seiyaku Kabushiki Kaisha

Pharmaceutical Co.,Ltd.

<120> HLA-A24-restricted cancer antigen peptide

<130>540886HT

<140>PCT/JP03/07463

<141>2003-06-12

<150>JP 2002-171518

<151>2002-06-12

<150>JP 2002-275572

<151>2002-09-20

<160>68

<210>1

<211>449

<212>PRT

<213> human

<400>1

Met Gly Ser Asp Val Arg Asp Leu Asn Ala Leu Leu Pro Ala Val Pro

1 5 10 15

Ser Leu Gly Gly Gly Gly Gly Cys Ala Leu Pro Val Ser Gly Ala Ala

20 25 30

Gln Trp Ala Pro Val Leu Asp Phe Ala Pro Pro Gly Ala Ser Ala Tyr

35 40 45

Gly Ser Leu Gly Gly Pro Ala Pro Pro Pro Ala Pro Pro Pro Pro Pro

50 55 60

Pro Pro Pro Pro His Ser Phe Ile Lys Gln Glu Pro Ser Trp Gly Gly

65 70 75 80

Ala Glu Pro His Glu Glu Gln Cys Leu Ser Ala Phe Thr Val His Phe

85 90 95

Ser Gly Gln Phe Thr Gly Thr Ala Gly Ala Cys Arg Tyr Gly Pro Phe

100 105 110

Gly Pro Pro Pro Pro Ser Gln Ala Ser Ser Gly Gln Ala Arg Met Phe

115 120 125

Pro Asn Ala Pro Tyr Leu Pro Ser Cys Leu Glu Ser Gln Pro Ala Ile

130 135 140

Arg Asn Gln Gly Tyr Ser Thr Val Thr Phe Asp Gly Thr Pro Ser Tyr

145 150 155 160

Gly His Thr Pro Ser His His Ala Ala Gln Phe Pro Asn His Ser Phe

165 170 175

Lys His Glu Asp Pro Met Gly Gln Gln Gly Ser Leu Gly Glu Gln Gln

180 185 190

Tyr Ser Val Pro Pro Pro Val Tyr Gly Cys His Thr Pro Thr Asp Ser

195 200 205

Cys Thr Gly Ser Gln Ala Leu Leu Leu Arg Thr Pro Tyr Ser Ser Asp

210 215 220

Asn Leu Tyr Gln Met Thr Ser Gln Leu Glu Cys Met Thr Trp Asn Gln

225 230 235 240

Met Asn Leu Cly Ala Thr Leu Lys Gly Val Ala Ala Gly Ser Ser Ser

245 250 255

Ser Val Lys Trp Thr Glu Gly Gln Ser Asn His Ser Thr Gly Tyr Glu

260 265 270

Ser Asp Asn His Thr Thr Pro Ile Leu Cys Gly Ala Gln Tyr Arg Ile

275 280 285

His Thr His Gly Val Phe Arg Gly Ile Gln Asp Val Arg Arg Val Pro

290 295 300

Gly Val Ala Pro Thr Leu Val Arg Ser Ala Ser Glu Thr Ser Glu Lys

305 310 315 320

Arg Pro Phe Met Cys Ala Tyr Pro Gly Cys Asn Lys Arg Tyr Phe Lys

325 330 335

Leu Ser His Leu Gln Met His Ser Arg Lys His Thr Gly Glu Lys Pro

340 345 350

Tyr Gln Cys Asp Phe Lys Asp Cys Glu Arg Arg Phe Ser Arg Ser Asp

355 360 365

Gln Leu Lys Arg His Gln Arg Arg His Thr Gly Val Lys Pro Phe Gln

370 375 380

Cys Lys Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu Lys Thr

385 390 395 400

His Thr Arg Thr His Thr Gly Lys Thr Ser Glu Lys Pro Phe Ser Cys

405 410 415

Arg Trp Pro Ser Cys Gln Lys Lys Phe Ala Arg Ser Asp Glu Leu Val

420 425 430

Arg His His Asn Met His Gln Arg Asn Met Thr Lys Leu Gln Leu Ala

435 440 445

Leu

<210>2

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>2

Arg Tyr Phe Pro Asn Ala Pro Tyr Leu

1 5

<210>3

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>3

Arg Tyr Pro Gly Val Ala Pro Thr Leu

1 5

<210>4

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>4

Arg Tyr Pro Ser Cys Gln Lys Lys Phe

1 5

<210>5

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>5

Ala Tyr Leu Pro Ala Val Pro Ser Leu

1 5

<210>6

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>6

Asn Tyr Met Asn Leu Gly Ala Thr Leu

1 5

<210>7

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>7

Arg Val Pro Gly Val Ala Pro Thr Leu

1 5

<210>8

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>8

Arg Met Phe Pro Asn Ala Pro Tyr Leu

1 5

<210>9

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>9

Arg Trp Pro Ser Cys Gln Lys Lys Phe

1 5

<210>10

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>10

Gln Tyr Arg Ile His Thr His Gly Val Phe

1 5 10

<210>11

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>11

Ala Tyr Pro Gly Cys Asn Lys Arg Tyr Phe

1 5 10

<210>12

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>12

Arg Tyr Phe Pro Asn Ala Pro Tyr Phe

1 5

<210>13

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>13

Arg Tyr Phe Pro Asn Ala Pro Tyr Trp

1 5

<210>14

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>14

Arg Tyr Phe Pro Asn Ala Pro Tyr Ile

1 5

<210>15

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>15

Arg Tyr Phe Pro Asn Ala Pro Tyr Met

1 5

<210>16

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>16

Arg Tyr Pro Gly Val Ala Pro Thr Phe

1 5

<210>17

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>17

Arg Tyr Pro Gly Val Ala Pro Thr Trp

1 5

<210>18

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>18

Arg Tyr Pro Gly Val Ala Pro Thr Ile

1 5

<210>19

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>19

Arg Tyr Pro Gly Val Ala Pro Thr Met

1 5

<210>20

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>20

Arg Tyr Pro Ser Cys Gln Lys Lys Trp

1 5

<210>21

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>21

Arg Tyr Pro Ser Cys Gln Lys Lys Leu

1 5

<210>22

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>22

Arg Tyr Pro Ser Cys Gln Lys Lys Ile

1 5

<210>23

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>23

Arg Tyr Pro Ser Cys Gln Lys Lys Met

1 5

<210>24

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>24

Ala Tyr Leu Pro Ala Val Pro Ser Phe

1 5

<210>25

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>25

Ala Tyr Leu Pro Ala Val Pro Ser Trp

1 5

<210>26

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>26

Ala Tyr Leu Pro Ala Val Pro Ser Ile

1 5

<210>27

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>27

Ala Tyr Leu Pro Ala Val Pro Ser Met

1 5

<210>28

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>28

Asn Tyr Met Asn Leu Gly Ala Thr Phe

1 5

<210>29

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>29

Asn Tyr Met Asn Leu Gly Ala Thr Trp

1 5

<210>30

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>30

Asn Tyr Met Asn Leu Gly Ala Thr Ile

1 5

<210>31

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>31

Asn Tyr Met Asn Leu Gly Ala Thr Met

1 5

<210>32

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>32

Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser

1 5 10 15

Ala Ser His Leu Glu

20

<210>33

<211>3857

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: the DNA region from position 1-1550 is from human, and the DNA region from position 1551-3857 is used for mouse

<400>33

aagcttactc tctggcacca aactccatgg gatgattttt cttctagaag agtccaggtg 60

gacaggtaag gagtgggagt cagggagtcc agttcaggga cagagattac gggatgaaaa 120

gtgaaaggag agggacgggg cccatgccga gggtttctcc cttgtttctc agacagctct 180

tgggccaaga ttcagggaga cattgagaca gagcgcttgg cacagaagca gaggggtcag 240

ggcgaagtcc cagggcccca ggcgtggctc tcagggtctc aggccccgaa ggcggtgtat 300

ggattgggga gtcccagcct tggggattcc ccaactccgc agtttctttt ctccctctcc 360

caacctatgt agggtccttc ttcctggata ctcacgacgc ggacccagtt ctcactccca 420

ttgggtgtcg ggtttccaga gaagccaatc agtgtcgtcg cggtcgctgt tctaaagtcc 480

gcacgcaccc accgggactc agattctccc cagacgccga ggatggccgt catggcgccc 540

cgaaccctcg tcctgctact ctcgggggcc ctggccctga cccagacctg ggcaggtgag 600

tgcggggtcg ggagggaaac ggcctctgcg gggagaagca aggggcccgc ctggcggggg 660

cgcaagaccc gggaagccgc gccgggagga gggtcgggcg ggtctcagcc actcctcgtc 720

cccaggctcc cactccatga ggtatttctc cacatccgtg tcccggcccg gccgcgggga 780

gccccgcttc atcgccgtgg gctacgtgga cgacacgcag ttcgtgcggt tcgacagcga 840

cgccgcgagc cagaggatgg agccgcgggc gccgtggata gagcaggagg ggccggagta 900

ttgggacgag gagacaggga aagtgaaggc ccactcacag actgaccgag agaacctgcg 960

gatcgcgctc cgctactaca accagagcga ggccggtgag tgaccccggc ccggggcgca 1020

ggtcacgacc cctcatcccc cacggacggg ccgggtcgcc cacagtctcc gggtccgaga 1080

tccaccccga agccgcggga ccccgagacc cttgccccgg gagaggccca ggcgccttaa 1140

cccggtttca ttttcagttt aggccaaaaa tccccccggg ttggtcgggg ccgggcgggg 1200

ctcgggggac tgggctgacc gcggggtcgg ggccaggttc tcacaccctc cagatgatgt 1260

ttggctgcga cgtggggtcg gacgggcgct tcctccgcgg gtaccaccag tacgcctacg 1320

acggcaagga ttacatcgcc ctgaaagagg acctgcgctc ttggaccgcg gcggacatgg 1380

cggctcagat caccaagcgc aagtgggagg cggcccatgt ggcggagcag cagagagcct 1440

acctggaggg cacgtgcgtg gacgggctcc gcagatacct ggagaacggg aaggagacgc 1500

tgcagcgcac gggtaccagg ggccacgggg cgcctacctg atcgcctgta gatcctgtgt 1560

gacacacctg taccttgtcc cccagagtca ggggctggga gtcattttct ctggctacac 1620

acttagtgat ggctgttcac ttggactgac agttaatgtt ggtcagcaag gtgactacaa 1680

tggttgagtc tcaatggtgt caccttccag gatcatacag ccctaatttt aatatgaact 1740

caaacacata ttaaattagt tattttccat tccctcctcc attctttgac tacctctctc 1800

atgctattga acatcacata aggatggcca tgtttaccca atggctcatg tggattccct 1860

cttagcttct gagtcccaaa agaaaatgtg cagtcctgtg ctgaggggac cagctctgct 1920

tttggtcact agtgcgatga cagttgaagt gtcaaacaga cacatagttc actgtcatca 1980

ttgatttaac tgagtcttgg gtagatttca gtttgtcttg ttaattgtgt gatttcttaa 2040

atcttccaca cagattcccc aaaggcccat gtgacccatc acagcagacc tgaagataaa 2100

gtcaccctga ggtgctgggc cctgggcttc taccctgctg acatcaccct gacctggcag 2160

ttgaatgggg aggagctgat ccaggacatg gagcttgtgg agaccaggcc tgcaggggat 2220

ggaaccttcc agaagtgggc atctgtggtg gtgcctcttg ggaaggagca gtattacaca 2280

tgccatgtgt accatcaggg gctgcctgag cccctcaccc tgagatgggg taaggagagt 2340

gtgggtgcag agctggggtc agggaaagct ggagctttct gcagaccctg agctgctcag 2400

ggctgagagc tggggtcatg accctcacct tcatttcttg tacctgtcct tcccagagcc 2460

tcctccatcc actgtctcca acatggcgac cgttgctgtt ctggttgtcc ttggagctgc 2520

aatagtcact ggagctgtgg tggcttttgt gatgaagatg agaaggagaa acacaggtag 2580

gaaagggcag agtctgagtt ttctctcagc ctcctttaga gtgtgctctg ctcatcaatg 2640

gggaacacag gcacacccca cattgctact gtctctaact gggtctgctg tcagttctgg 2700

gaacttccta gtgtcaagat cttcctggaa ctctcacagc ttttcttctc acaggtggaa 2760

aaggagggga ctatgctctg gctccaggtt agtgtgggga cagagttgtc ctggggacat 2820

tggagtgaag ttggagatga tgggagctct gggaatccat aatagctcct ccagagaaat 2880

cttctaggtg cctgagttgt gccatgaaat gaatatgtac atgtacatat gcatatacat 2940

ttgttttgtt ttaccctagg ctcccagacc tctgatctgt ctctcccaga ttgtaaaggt 3000

gacactctag ggtctgattg gggaggggca atgtggacat gattgggttt caggaactcc 3060

cagaatcccc tgtgagtgag tgatgggttg ttcgaatgtt gtcttcacag tgatggttca 3120

tgaccctcat tctctagcgt gaagacagct gcctggagtg gacttggtga cagacaatgt 3180

cttctcatat ctcctgtgac atccagagcc ctcagttctc tttagtcaag tgtctgatgt 3240

tccctgtgag cctatggact caatgtgaag aactgtggag cccagtccac ccctctacac 3300

caggaccctg tccctgcact gctctgtctt cccttccaca gccaaccttg ctggttcagc 3360

caaacactga gggacatctg tagcctgtca gctccatgct accctgacct gcaactcctc 3420

acttccacac tgagaataat aatttgaatg taaccttgat tgttatcatc ttgacctagg 3480

gctgatttct tgttaatttc atggattgag aatgcttaga ggttttgttt gtttgtttga 3540

ttgatttgtt tttttgaaga aataaatgat agatgaataa acttccagaa tctgggtcac 3600

tatgctgtgt gtatctgttg ggacaggatg agactgtagc agctgagtgt gaacagggct 3660

gtgccgaggt gggctcagtt tgctttgatc tgtgatgggg ccacacctcc actgtgtcac 3720

ctctgggctc tgttccctct atcactatga ggcacatgct gagagtttgt ggtcacaaag 3780

acacagggaa ggcctgagcc ttgccctgtc cccaggatta tgagccccca gggctaaaga 3840

tcagagactc ggaattc 3857

<210>34

<211>1119

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: the DNA region from positions 1-618 was from human, and the DNA region from position 619-1119 was from mouse

<400>34

atg gcc gtc atg gcg ccc cga acc ctc gtc ctg cta ctc tcg ggg gcc 48

Met Ala Val Met Ala Pro Arg Thr Leu Val Leu Leu Leu Ser Gly Ala

5 10 15

ctg gcc ctg acc cag acc tgg gca ggc tcc cac tcc atg agg tat ttc 96

Leu Ala Leu Thr Gln Thr Trp Ala Gly Ser His Ser Met Arg Tyr Phe

20 25 30

tcc aca tcc gtg tcc cgg ccc ggc cgc ggg gag ccc cgc ttc atc gcc 144

Ser Thr Ser Val Ser Arg Pro Gly Arg Gly Glu Pro Arg Phe Ile Ala

35 40 45

gtg ggc tac gtg gac gac acg cag ttc gtg cgg ttc gac agc gac gcc 192

Val Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp Ser Asp Ala

50 55 60

gcg agc cag agg atg gag ccg cgg gcg ccg tgg ata gag cag gag ggg 240

Ala Ser Gln Arg Met Glu Pro Arg Ala Pro Trp Ile Glu Gln Glu Gly

65 70 75 80

ccg gag tat tgg gac gag gag aca ggg aaa gtg aag gcc cac tca cag 288

Pro Glu Tyr Trp Asp Glu Glu Thr Gly Lys Val Lys Ala His Ser Gln

85 90 95

act gac cga gag aac ctg cgg atc gcg ctc cgc tac tac aac cag agc 336

Thr Asp Arg Glu Asn Leu Arg Ile Ala Leu Arg Tyr Tyr Asn Gln Ser

100 105 110

gag gcc ggt tct cac acc ctc cag atg atg ttt ggc tgc gac gtg ggg 384

Glu Ala Gly Ser His Thr Leu Gln Met Met Phe Gly Cys Asp Val Gly

115 120 125

tcg gac ggg cgc ttc ctc cgc ggg tac cac cag tac gcc tac gac ggc 432

Ser Asp Gly Arg Phe Leu Arg Gly Tyr His Gln Tyr Ala Tyr Asp Gly

130 135 140

aag gat tac atc gcc ctg aaa gag gac ctg cgc tct tgg acc gcg gcg 480

Lys Asp Tyr Ile Ala Leu Lys Glu Asp Leu Arg Ser Trp Thr Ala Ala

145 150 155 160

gac atg gcg gct cag atc acc aag cgc aag tgg gag gcg gcc cat gtg 528

Asp Met Ala Ala Gln Ile Thr Lys Arg Lys Trp Glu Ala Ala His Val

165 170 175

gcg gag cag cag aga gcc tac ctg gag ggc acg tgc gtg gac ggg ctc 576

Ala Glu Gln Gln Arg Ala Tyr Leu Glu Gly Thr Cys Val Asp Gly Leu

180 185 190

cgc aga tac ctg gag aac ggg aag gag acg ctg cag cgc acg gat tcc 624

Arg Arg Tyr Leu Glu Asn Gly Lys Glu Thr Leu Gln Arg Thr Asp Ser

195 200 205

cca aag gcc cat gtg acc cat cac agc aga cct gaa gat aaa gtc acc 672

Pro Lys Ala His Val Thr His His Ser Arg Pro Glu Asp Lys Val Thr

210 215 220

ctg agg tgc tgg gcc ctg ggc ttc tac cct gct gac atc acc ctg acc 720

Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Asp Ile Thr Leu Thr

225 230 235 240

tgg cag ttg aat ggg gag gag ctg atc cag gac atg gag ctt gtg gag 768

Trp Gln Leu Asn Gly Glu Glu Leu Ile Gln Asp Met Glu Leu Val Glu

245 250 255

acc agg cct gca ggg gat gga acc ttc cag aag tgg gca tct gtg gtg 816

Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ser Val Val

260 265 270

gtg cct ctt ggg aag gag cag tat tac aca tgc cat gtg tac cat cag 864

Val Pro Leu Gly Lys Glu Gln Tyr Tyr Thr Cys His Val Tyr His Gln

275 280 285

ggg ctg cct gag ccc ctc acc ctg aga tgg gag cct cct cca tcc act 912

Gly Leu Pro Glu Pro Leu Thr Leu Arg Trp Glu Pro Pro Pro Ser Thr

290 295 300

gtc tcc aac atg gcg acc gtt gct gtt ctg gtt gtc ctt gga gct gca 960

Val Ser Asn Met Ala Thr Val Ala Val Leu Val Val Leu Gly Ala Ala

305 310 315 320

ata gtc act gga gct gtg gtg gct ttt gtg atg aag atg aga agg aga 1008

Ile Val Thr Gly Ala Val Val Ala Phe Val Met Lys Met Arg Arg Arg

325 330 335

aac aca ggt gga aaa gga ggg gac tat gct ctg gct cca ggc tcc cag 1056

Asn Thr Gly Gly Lys Gly Gly Asp Tyr Ala Leu Ala Pro Gly Ser Gln

340 345 350

acc tct gat ctg tct ctc cca gat tgt aaa gtg atg gtt cat gac cct 1104

Thr Ser Asp Leu Ser Leu Pro Asp Cys Lys Val Met Val His Asp Pro

355 360 365

cat tct cta gcg tga 1119

His Ser Leu Ala

370

<210>35

<211>372

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: the polypeptide from positions 1-206 is from human, and the polypeptide region from position 207-372 is from mouse

<400>35

Met Ala Val Met Ala Pro Arg Thr Leu Val Leu Leu Leu Ser Gly Ala

5 10 15

Leu Ala Leu Thr Gln Thr Trp Ala Gly Ser His Ser Met Arg Tyr Phe

20 25 30

Ser Thr Ser Val Ser Arg Pro Gly Arg Gly Glu Pro Arg Phe Ile Ala

35 40 45

Val Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp Ser Asp Ala

50 55 60

Ala Ser Gln Arg Met Glu Pro Arg Ala Pro Trp Ile Glu Gln Glu Gly

65 70 75 80

Pro Glu Tyr Trp Asp Glu Glu Thr Gly Lys Val Lys Ala His Ser Gln

85 90 95

Thr Asp Arg Glu Asn Leu Arg Ile Ala Leu Arg Tyr Tyr Asn Gln Ser

100 105 110

Glu Ala Gly Ser His Thr Leu Gln Met Met Phe Gly Cys Asp Val Gly

115 120 125

Ser Asp Gly Arg Phe Leu Arg Gly Tyr His Gln Tyr Ala Tyr Asp Gly

130 135 140

Lys Asp Tyr Ile Ala Leu Lys Glu Asp Leu Arg Ser Trp Thr Ala Ala

145 150 155 160

Asp Met Ala Ala Gln Ile Thr Lys Arg Lys Trp Glu Ala Ala His Val

165 170 175

Ala Glu Gln Gln Arg Ala Tyr Leu Glu Gly Thr Cys Val Asp Gly Leu

180 185 190

Arg Arg Tyr Leu Glu Asn Gly Lys Glu Thr Leu Gln Arg Thr Asp Ser

195 200 205

Pro Lys Ala His Val Thr His His Ser Arg Pro Glu Asp Lys Val Thr

210 215 220

Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Asp Ile Thr Leu Thr

225 230 235 240

Trp Gln Leu Asn Gly Glu Glu Leu Ile Gln Asp Met Glu Leu Val Glu

245 250 255

Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala Ser Val Val

260 265 270

Val Pro Leu Gly Lys Glu Gln Tyr Tyr Thr Cys His Val Tyr His Gln

275 280 285

Gly Leu Pro Glu Pro Leu Thr Leu Arg Trp Glu Pro Pro Pro Ser Thr

290 295 300

Val Ser Asn Met Ala Thr Val Ala Val Leu Val Val Leu Gly Ala Ala

305 310 315 320

Ile Val Thr Gly Ala Val Val Ala Phe Val Met Lys Met Arg Arg Arg

325 330 335

Asn Thr Gly Gly Lys Gly Gly Asp Tyr Ala Leu Ala Pro Gly Ser Gln

340 345 350

Thr Ser Asp Leu Ser Leu Pro Asp Cys Lys Val Met Val His Asp Pro

355 360 365

His Ser Leu Ala

370

<210>36

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>36

cccaagctta ctctctggca ccaaactcca tgggat 36

<210>37

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>37

cgggagatct acaggcgatc aggtaggcgc 30

<210>38

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>38

cgcaggctct cacactattc aggtgatctc 30

<210>39

<211>38

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>39

cggaattccg agtctctgat ctttagccct gggggctc 38

<210>40

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>40

aggacttgga ctctgagagg cagggtctt 29

<210>41

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>41

catagtcccc tccttttcca cctgtgagaa 30

<210>42

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>42

cgaaccctcg tcctgctact ctc 23

<210>43

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>43

agcatagtcc cctccttttc cac 23

<210>44

<211>39

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>44

cccaagcttc gccgaggatg gccgtcatgg cgccccgaa 39

<210>45

<211>41

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>45

ccggaattct gtcttcacgc tagagaatga gggtcatgaa c 41

<210>46

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>46

Pro Tyr Val Ser Arg Leu Leu Gly Ile

5

<210>47

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>47

Ile Met Pro Lys Ala Gly Leu Leu Ile

5

<210>48

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>48

Thr Tyr Ala Cys Phe Val Ser Asn Leu

5

<210>49

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400>49

Gln Tyr Ser Trp Phe Val Asn Gly Thr Phe

5 10

<210>50

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>50

Ala Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu

1 5 10 15

<210>51

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>51

Ala Leu Leu Pro Ala Val Pro Ser Leu

1 5

<210>52

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>52

Asn Gln Met Asn Leu Gly Ala Thr Leu

1 5

<210>53

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>53

Arg Phe Phe Pro Asn Ala Pro Tyr Leu

1 5

<210>54

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>54

Arg Trp Phe Pro Asn Ala Pro Tyr Leu

1 5

<210>55

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>55

Arg Phe Pro Gly Val Ala Pro Thr Leu

1 5

<210>56

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>56

Arg Met Pro Gly Val Ala Pro Thr Leu

1 5

<210>57

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>57

Arg Trp Pro Gly Val Ala Pro Thr Leu

1 5

<210>58

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>58

Arg Phe Pro Ser Cys Gln Lys Lys Phe

1 5

<210>59

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>59

Arg Met Pro Ser Cys Gln Lys Lys Phe

1 5

<210>60

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>60

Ala Phe Leu Pro Ala Val Pro Ser Leu

1 5

<210>61

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>61

Ala Met Leu Pro Ala Val Pro Ser Leu

1 5

<210>62

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>62

Ala Trp Leu Pro Ala Val Pro Ser Leu

1 5

<210>63

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>63

Asn Phe Met Asn Leu Gly Ala Thr Leu

1 5

<210>64

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>64

Asn Met Met Asn Leu Gly Ala Thr Leu

1 5

<210>65

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>65

Asn Trp Met Asn Leu Gly Ala Thr Leu

1 5

<210>66

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>66

Arg Tyr Pro Ser Ser Gln Lys Lys Phe

1 5

<210>67

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>67

Arg Tyr Pro Ser Ala Gln Lys Lys Phe

1 5

<210>68

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<223> Xaa at position 5 represents Abu

<400>68

Arg Tyr Pro Ser Xaa Gln Lys Lys Phe

1 5

Claims (11)

1. A peptide consisting of the amino acid sequence of Arg Tyr Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO: 2).

2. A polynucleotide encoding the peptide of claim 1.

3. An expression vector comprising the polynucleotide of claim 2.

4. A cell comprising the expression vector of claim 3.

5. A method of making the peptide of claim 1, comprising culturing the cell of claim 4 under conditions suitable for expression of the peptide.

6. An antibody that specifically binds to the peptide of claim 1.

7. An antigen-presenting cell that presents on its surface a complex formed between the peptide of claim 1 and HLA-a24 antigen.

8. A Cytotoxic T Lymphocyte (CTL) that recognizes a complex formed between the peptide according to claim 1 and the HLA-a24 antigen.

9. A pharmaceutical composition comprising the peptide of claim 1, the polynucleotide of claim 2, the expression vector of claim 3, the cell of claim 4, the antigen presenting cell of claim 7, or the cytotoxic T lymphocyte of claim 8, together with a pharmaceutically acceptable carrier.

10. A cancer vaccine comprising the peptide of claim 1, the polynucleotide of claim 2, the expression vector of claim 4, the cell of claim 4, the antigen presenting cell of claim 7, or the cytotoxic T lymphocyte of claim 8 as an active ingredient.

11. Use of the peptide of claim 1, the polynucleotide of claim 2, the expression vector of claim 3, the cell of claim 4, the antigen presenting cell of claim 7, or the cytotoxic T lymphocyte of claim 8 in the preparation of a cancer vaccine.